CLEC5A-Mediated Enhancement of the Inflammatory Response in Myeloid Cells Contributes to Influenza Virus Pathogenicity In Vivo

Abstract

Human infections with influenza viruses exhibit mild to severe clinical outcomes as a result of complex virus-host interactions. Induction of inflammatory mediators via pattern recognition receptors may dictate subsequent host responses for pathogen clearance and tissue damage. We identified that human C-type lectin domain family 5 member A (CLEC5A) interacts with the hemagglutinin protein of influenza viruses expressed on lentiviral pseudoparticles through lectin screening. Silencing CLEC5A gene expression, blocking influenza-CLEC5A interactions with anti-CLEC5A antibodies, or dampening CLEC5A-mediated signaling using a spleen tyrosine kinase inhibitor consistently reduced the levels of proinflammatory cytokines produced by human macrophages without affecting the replication of influenza A viruses of different subtypes. Infection of bone marrow-derived macrophages from CLEC5A-deficient mice showed reduced levels of tumor necrosis factor alpha (TNF-α) and IP-10 but elevated alpha interferon (IFN-α) compared to those of wild-type mice. The heightened type I IFN response in the macrophages of CLEC5A-deficient mice was associated with upregulated TLR3 mRNA after treatment with double-stranded RNA. Upon lethal challenges with a recombinant H5N1 virus, CLEC5A-deficient mice showed reduced levels of proinflammatory cytokines, decreased immune cell infiltration in the lungs, and improved survival compared to the wild-type mice, despite comparable viral loads noted throughout the course of infection. The survival difference was more prominent at a lower dose of inoculum. Our results suggest that CLEC5A-mediated enhancement of the inflammatory response in myeloid cells contributes to influenza pathogenicity in vivo and may be considered a therapeutic target in combination with effective antivirals. Well-orchestrated host responses together with effective viral clearance are critical for optimal clinical outcome after influenza infections.

Importance: Multiple pattern recognition receptors work in synergy to sense viral RNA or proteins synthesized during influenza replication and mediate host responses for viral control. Well-orchestrated host responses may help to maintain the inflammatory response to minimize tissue damage while inducing an effective adaptive immune response for viral clearance. We identified that CLEC5A, a C-type lectin receptor which has previously been reported to mediate flavivirus-induced inflammatory responses, enhanced induction of proinflammatory cytokines and chemokines in myeloid cells after influenza infections. CLEC5A-deficient mice infected with influenza virus showed reduced inflammation in the lungs and improved survival compared to that of the wild-type mice despite comparable viral loads. The survival difference was more prominent at a lower dose of inoculum. Collectively, our results suggest that dampening CLEC5A-mediated inflammatory responses in myeloid cells reduces immunopathogenesis after influenza infections.

Keywords: C-type lectins; CLEC5A; influenza virus; macrophages; spleen tyrosine kinase (Syk).

FIG 1

Identification of CLEC5A as potential PRR for influenza virus. The relative binding intensity of the pseudoparticles expressing influenza HA derived from H5N1 or H1N1 viruses to a panel of soluble human CLRs is shown. Briefly, 0.5 μg of soluble human CLRs was coated on the plate, followed by incubation with the HA-expressing pseudoparticles. The bound pseudoparticles were detected by anti-H5 or anti-H1 antibodies. The absorbance (Abs) obtained was normalized to the absorbance of CLEC4L. The dotted line represents 50% binding signal compared to that of CLEC4L. The data shown are the means ± standard deviations (SD) from 8 replicates from 4 independent experiments.

FIG 2

CLEC5A mediates enhanced proinflammatory cytokine and chemokine induction in human M-Mϕ after influenza virus infection. M-Mϕ differentiated from the PBMC of 8 independent donors were transfected with CLEC5A gene-specific or nontargeting siRNA, followed by cell sorting to obtain the CLEC5A-positive (CLEC5A_pos) and CLEC5A-negative (CLEC5A_neg) populations. The macrophages were then infected with HK^HA,NA or VN^HA,NA recombinant viruses at an MOI of 2 for 24 h to determine viral M gene copy numbers in infected M-Mϕ and viral titers in culture supernatants (log₁₀ TCID₅₀/ml in MDCK cells) (A) and proinflammatory cytokines and chemokines in culture supernatant (means ± SD, in pg/ml) (B). P values from Mann-Whitney tests are shown.

FIG 3

CLEC5A mediates enhanced proinflammatory cytokine and chemokine induction in human GM-Mϕ after influenza virus infection. GM-Mϕ differentiated from the PBMC of 2 independent donors were transfected with CLEC5A gene-specific or nontargeting siRNA, followed by cell sorting to obtain the CLEC5A-positive (CLEC5A_pos) and CLEC5A-negative (CLEC5A_neg) populations. The macrophages were then infected with HK^HA,NA or VN^HA,NA recombinant viruses at an MOI of 2 for 24 h to determine viral M gene copy numbers in infected GM-Mϕ and viral titers in culture supernatant (log₁₀ TCID₅₀/ml in MDCK cells) (A) and proinflammatory cytokines and chemokines in culture supernatants (means ± SD, in pg/ml) (B). P values from Mann-Whitney tests are shown.

FIG 4

CLEC5A-mediated proinflammatory response in human M-Mϕ after infections with influenza viruses of H1N1, H5N1, and H7N9 subtypes. Human M-Mϕ differentiated from PBMC of 2 independent donors were infected with A/Hong Kong/54/98 (H1N1), A/Vietnam/1203/04 (H5N1), or A/Shanghai/2/13 (H7N9) influenza viruses at an MOI of 2 for 24 h to determine viral M gene copy numbers in infected M-Mϕ (A) and proinflammatory cytokines and chemokines in culture supernatants (means ± SD, in pg/ml) (B). P values from Mann-Whitney tests are shown.

FIG 5

Blocking CLEC5A-mediated signaling with Syk inhibitor (Bay 61-3606) and CLEC5A antagonizing antibodies reduced inflammatory response in human M-Mϕ after influenza virus infection. (A) Human M-Mϕ differentiated from the PBMC of 3 independent donors were treated with dimethyl sulfoxide or the Syk inhibitor Bay 61-3606 prior to and throughout the course of infection with HK^HA,NA or VN^HA,NA virus at an MOI of 2 to determine the TNF-α and IP-10 concentrations (means ± SD, in pg/ml) from culture supernatants at 24 h postinfection. (B) Human M-Mϕ differentiated from the PBMC of 4 independent donors were incubated with 1 μg/ml of murine IgG1 isotype control antibody (Iso ctrl) or clones of anti-human CLEC5A antibodies, which were previously shown to possess antagonizing activity against DV or JEV infections, prior to and throughout the course of infection with VN^HA,NA virus at an MOI of 2 to determine the level of IP-10 (means ± SD, in pg/ml) from culture supernatants at 24 h postinfection. (C) Human M-Mϕ differentiated from the PBMC of 4 independent donors were incubated with increasing concentrations of murine IgG1 isotype control antibody or the anti-CLEC5A antibody (clone 8H8F5) prior to and throughout the course of infection with VN^HA,NA virus at an MOI of 2 to determine the level of IP-10 (means ± SD, in pg/ml) from culture supernatants at 24 h postinfection. ns, not significant. (D) M-Mϕ differentiated from the PBMC of 2 independent donors were transfected with CLEC5A gene-specific or nontargeting siRNA followed by cell sorting to obtain the CLEC5A-positive (CLEC5A_pos) and CLEC5A-negative (CLEC5A_neg) populations. M-Mϕ were infected with VN^HA,NA or UV-inactivated VN^HA,NA (UV-VN^HA,NA) at an MOI of 2. Viral M gene copy numbers and levels of TNF-α and IP-10 (means ± SD, in pg/ml) in culture supernatant were determined at 24 h postinfection. P values from Mann-Whitney tests are shown.

FIG 6

(A) Inflammatory response in BMM derived from CLEC5A^−/− or WT mice after incubation with TLR agonists or after influenza infection. BMM derived from CLEC5A^−/− or WT mice were treated with gardiquimod (GRD; 10 μg/ml), lipopolysaccharide (LPS; 100 ng/ml), poly(I·C) (PIC; 50 μg/ml), or zymosan (Zy; 10 μg/ml) for 24 h to determine the level of cytokines and chemokines in the supernatant (means ± SD, in pg/ml, derived from 6 replicates of BMM obtained from 2 mice). (B) BMM derived from CLEC5A^−/− or WT mice were infected with VN^HA,NA at an MOI of 2 for 24 h to determine the levels of cytokines and chemokines in the supernatant (means ± SD, in pg/ml, derived from 9 replicates of BMM obtained from 3 mice). (C) Copy number of TLR3 mRNA in BMM after activation with TLR agonist or influenza infection at an MOI of 2 for 24 h (means ± SD from 4 replicates of BMM obtained from 2 mice). P values from Mann-Whitney tests are shown.

FIG 7

Lower levels of proinflammatory cytokines and reduced level of immune cell infiltration in the lungs of CLEC5A^−/− mice compared to the WT mice after lethal challenge of 5 MLD₅₀ of VN^HA,NA virus. WT and CLEC5A^−/− mice were infected with 5 MLD₅₀ of VN^HA,NA intranasally; mouse lungs were collected at days 1, 4, and 7 postinfection (n = 5 or 6 per group at each time point) to determine the concentrations of cytokines and chemokines (means ± SD, in pg/ml) (A) and virus titers (means ± SD, in log₁₀ TCID₅₀/ml) (B). (C and D) BAL fluid was collected from WT and CLEC5A^−/− mice (n = 10 per group at each time point) to monitor the total cell count (means ± SD) detected on days 3 and 7 postinfection (C) and the immunophenotyping result on day 7 postinfection using surface markers (P values from Mann-Whitney tests are shown) (D). Hematoxylin and eosin staining of the CLEC5A^−/− (E) and WT (F) mouse lungs at day 7 postinfection with 5 MLD₅₀ of VN^HA,NA (n = 2 per group). (G and H) Changes in body weight (G) and survival (H) of WT and CLEC5A^−/− mice after challenge with 5 MLD₅₀ (P value from log-rank test is shown).

FIG 8

CLEC5A^−/− mice were protected from the sublethal challenge of VN^HA,NA virus by reducing the level of proinflammatory cytokine in the lungs. WT and CLEC5A^−/− mice were infected with 1 MLD₅₀ of VN^HA,NA intranasally. Mouse survival (A) and body weight changes (B) were monitored every other day; P values from log-rank tests are shown. (C and D) Viral titer (means ± SD, in log₁₀ TCID₅₀/ml) (C) and cytokine and chemokine levels (means ± SD, in pg/ml) (D) detected in the mouse lungs at day 1, 4, and 7 postinfection (n = 4 per group at each time point); P values from Mann-Whitney tests are shown.