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.

enza 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, spleen tyrosine kinase (Syk), influenza virus, macrophages I nfluenza viruses impact human health through annual epidemics, intermittent pandemics, and sporadic zoonotic infections. Infection may lead to self-limited or severe clinical symptoms as a result of complex virus-host interactions (1)(2)(3)(4)(5). Activation of pattern recognition receptors (PRRs) that rapidly respond to invading pathogens or damage-associated molecular patterns (DAMP) through induction of inflammatory mediators may dictate the subsequent adaptive immune response for pathogen clearance and clinical outcome. Various intracellular PRRs, including the Toll-like receptors (TLR) 3, 7, and 10 (6-9), retinoic acid-inducible gene I (RIG-I) (10), or Nod-like receptors NLRP3 and NOD2 (11)(12)(13), sense viral RNA or proteins produced during influenza virus replication, leading to induction of an inflammatory response followed by activation of CD4 ϩ or CD8 ϩ T cells (5,14). Poorly coordinated host responses may pave the way for severe clinical outcomes characterized by elevated inflammatory responses, increased leukocyte infiltration, and pulmonary tissue injury (15)(16)(17)(18)(19).
Signaling through C-type lectin receptors (CLRs) may further modulate host defense against infections by different microbes (20). The C-type lectin superfamily includes soluble or integral proteins containing the characteristic C-type lectin-like domain (CTLD) that binds to carbohydrates, lipids, or proteins (21). The integral CLRs on myeloid cells can signal through the immunoreceptor tyrosine-based activation motif (ITAM) or immunoreceptor tyrosine-based inhibition motif (ITIM) signaling motifs either directly or in association with other adaptor proteins (21,22) and trigger a plethora of myeloid cell responses, such as phagocytosis (23), priming cytotoxic T lymphocytes (24), respiratory burst (25), or production of proinflammatory cytokines (26,27). Furthermore, CLR signaling may antagonize or synergize the signals from other PRRs to regulate the host immune response (21,22).
Multiple soluble C-type lectins, such as mannose-binding lectin (MBL), surfactant protein D (SP-D), and collectin kidney 1, possess neutralizing activity against influenza virus during infection (28)(29)(30). Galectin-1 of the S-type lectin family and the serum amyloid P of the pentraxin family have also been shown to ameliorate influenza infectivity by inhibiting hemagglutinin (HA) activity (31,32). In addition, CLRs expressed on myeloid cells, including DC-SIGN, DC-SIGNR, macrophage mannose receptor (MMR), and macrophage galactose-type lectin (MGL), can mediate influenza virus internalization in a sialic acid-independent manner (33)(34)(35)(36). However, it is not known if influenza infections trigger signaling via myeloid CLRs that further modulate the host immune response. Here, we identify that the spleen tyrosine kinase-coupled C-type lectin domain family 5 member A (CLEC5A), which has previously been reported to mediate flavivirus-induced inflammatory response (26,27,37), enhanced induction of proinflammatory cytokines and chemokines in myeloid cells after influenza A virus infection. Blockade of CLEC5A-mediated signaling attenuated influenza virus-induced inflammatory responses in macrophages without affecting viral replication. With reduced cytokines but comparable viral loads in mouse lungs, the CLEC5A knockout (CLEC5A Ϫ/Ϫ ) mice were protected after lethal influenza challenge compared to C57BL/6 wild-type (WT) mice, although a more prominent difference was observed when they were challenged with a lower inoculum. This suggested that dampening CLEC5A-mediated inflammatory responses in the myeloid cells is able to alleviate influenza pathogenicity in vivo. Our results highlight the significance of viral load control and a wellorchestrated host response in influenza pathogenesis.

RESULTS
Binding of influenza HA-expressing pseudotyped lentiviral particles to human soluble CLEC5A. The hemagglutinin (HA) glycoproteins are abundantly present on the surface of influenza virions and have been reported to interact with different CLRs to facilitate viral entry through glycans synthesized during posttranslational modifications (33)(34)(35)(36). Differential inflammatory cytokine responses have been reported after infections with H5N1 and H1N1 influenza viruses (16,38,39). To investigate the potential interactions between influenza virus and human CLRs, pseudotyped lentiviral particles with surface expression of influenza HA proteins derived from an H5N1 isolate, A/Vietnam/1203/04 (PP-VN HA ), or a seasonal H1N1 isolate, A/Hong Kong/54/98 (PP-HK HA ), were coincubated with a panel of soluble recombinant human CLR-Fc fusion proteins precoated on 96-well plates. The functions of the HA proteins expressed by PP-VN HA and PP-HK HA resemble the biological functions of those presented by influenza viruses with hemagglutination activity to turkey red blood cells, and they mediate entry and the transduction of renilla luciferase reporter in MDCK cells (data not shown). Pseudoparticles without surface glycoprotein expression were coincubated with soluble recombinant human CLR-Fc fusion proteins as negative controls to determine the background binding signals. The binding intensity of the HA-expressing pseudoparticles to each CLR was normalized to the binding intensity to CLEC4L (DC-SIGN), which is known to interact with influenza HA (33) (Fig. 1). CLEC4M (DC-SIGNR), CLEC7A, CLEC4C, CLEC9A, and CLEC5A gave more than 50% binding signal relative to that of CLEC4L for PP-VN HA,NA . The detection of a positive signal indicates that there is either a direct interaction between the soluble CLR and the HA-expressing pseudoparticles or that there is an indirect mediator bound to both the soluble CLR and the HA-expressing pseudoparticles. For example, DC-SIGN is known to recognize the high-mannose N-linked glycans present on influenza HA glycoprotein (33). CLEC5A was selected for further investigation, as it gave the highest binding signal to the PP-VN HA virus expressing H5 HA protein. CLEC5A activation by dengue virus (DV) or Japanese encephalitis virus (JEV) induces DAP12 phosphorylation and mediates induction of proinflammatory cytokines (tumor necrosis factor alpha [TNF-␣] and interleukin-6 [IL-6]) and chemokines (IP-10, MIP-1␣, monocyte chemoattractant protein 1 [MCP-1], and IL-8) (26,37,40). Since hyperinduction of proinflammatory cytokines is a signature for H5N1 pathogenesis (16), we hypothesized that CLEC5A is involved in regulating the host inflammatory response upon influenza virus infection.
CLEC5A enhanced proinflammatory response in human PBMC-derived macrophages after influenza virus infection. To study the CLEC5A-mediated host response, human peripheral blood mononuclear cells (PBMCs) obtained from four donors were each differentiated into macrophages and dendritic cells, and CLEC5A surface expression was monitored. High levels of CLEC5A were detected on CD14 ϩ / major histocompatibility complex class II-positive (MHC-II ϩ ) macrophages differentiated with macrophage colony-stimulating factor (M-CSF) (M-M) (72.9% Ϯ 8.9%) or granulocyte-macrophage colony-stimulating factor (GM-CSF) (GM-M) (94.8% Ϯ 4.9%) but not on those differentiated with autologous serum (17.2% Ϯ 11.4%) or on the CD14 Ϫ /MHC-II ϩ dendritic cells differentiated with GM-CSF and IL-4 (11.9% Ϯ 9.0%) (data not shown). Short interfering RNA (siRNA)-mediated gene silencing was applied to knock down CLEC5A expression in M-M and GM-M. Among the primary macrophages transfected with nontargeting control siRNA at 72 h posttransfection, 89.6% of cells expressed CLEC5A on the cell surface; in comparison, 71.1% of the primary macrophages transfected with the siRNA targeting human CLEC5A expressed CLEC5A. Due to the inefficient siRNA knockdown efficiency in primary macrophages, we proceeded to cell sorting after siRNA knockdown to yield CLEC5A ϩ and CLEC5A Ϫ populations as described in Materials and Methods. Specifically, at 72 h after siRNA knockdown followed by negative cell sorting, 79.4% of the M-M transfected with nontargeting control siRNA (siNT) and 15.6% of the M-M transfected with CLCE5Aspecific siRNA (siCLEC5A) showed surface expression of CLEC5A. We observed that the surface expression of CLEC5A was lowest at 72 h posttransfection and continued to remain low at 96 h posttransfection (data not shown). As such, infections with influenza viruses were performed at 72 h posttransfection with samples collected 24 h later (96 h posttransfection).
Previous studies from our laboratory have demonstrated differential proinflammatory cytokine responses in human macrophages after infections with A/Vietnam/ 1203/04 (H5N1) and A/HK/54/98 (H1N1) influenza viruses (38,39). We hypothesized that such differential cytokine responses are induced through interactions between influenza surface glycoprotein and CLEC5A. The  (Fig. 4B). Overall, these results suggest that CLEC5A mediates proinflammatory cytokine and chemokine induction in human PBMC-derived macrophages independent of the influenza virus subtypes.
Blocking CLEC5A-mediated signaling reduced inflammatory response in M-M after influenza infection. CLEC5A mediates inflammatory responses through association with the ITAM-containing DAP12 or the YINM-containing DAP10 adaptor proteins, followed by spleen tyrosine kinase (Syk) or phosphoinositide 3-kinase (PI3K)-mediated signaling pathways (22,26,27,40,41). Since Syk-mediated signaling was critical for CLEC5A-induced lethal shock in mice (41) and inflammasome activation in DV pathogenesis (27) (Fig. 5D). Similar results were observed when the MOI was increased from 2 to 20 (data not shown). This observation is in accord with our previous observation that UV-inactivated DV is less potent than live DV to activate CLEC5A (26), and active viral replication is required for the CLEC5A-mediated enhancement of the inflammatory response in M-M.
Influenza infections in bone marrow-derived macrophages of CLEC5A ؊/؊ mice showed reduced levels of TNF-␣ and IP-10 but increased IFN-␣ compared WT mice. CLEC5A Ϫ/Ϫ mice with deletion of exons 3 to 5 of the murine Clec5a genomic DNA (37) and the genetically matched WT mice were applied to evaluate the impact of CLEC5A on influenza pathogenesis. We first characterized the inflammatory response of bone marrow-derived macrophages (BMM) from CLEC5A Ϫ/Ϫ or WT mice after treatment with zymosan (Zy; TLR2 agonist), poly(I·C) (PIC; TLR3 agonist), lipopolysaccharide (LPS; TLR4 agonist), or gardiquimod (GRD; TLR7 agonist). The levels of TNF-␣ were comparable between BMM derived from CLEC5A Ϫ/Ϫ and WT mice after treatment with PIC, LPS, GRD, or Zy (Fig. 6A). However, BMM from CLEC5A Ϫ/Ϫ mice showed an increased type I interferon response compared to that of the WT mice after PIC treatment (Fig. 6A). In addition, reduced IP-10 was detected from the macrophages derived from the CLEC5A Ϫ/Ϫ mice after treatment with PIC or Zy.
BMM derived from CLEC5A Ϫ/Ϫ or WT mice were infected with VN HA,NA virus at an MOI of 2, and the levels of cytokines and chemokines from the culture supernatant were determined at 24 h postinfection. The VN HA,NA virus showed comparable infectivity in BMM derived from CLEC5A Ϫ/Ϫ or WT mice. As observed in the CLEC5A Ϫ human M-M, reduced levels of TNF-␣ and IP-10 (P ϭ 0.063 and P ϭ 0.046, respectively) were detected in BMM of CLEC5A Ϫ/Ϫ mice compared to those in the WT mice after infection (Fig. 6B). Interestingly, a higher level of IFN-␣ was detected in the CLEC5A Ϫ/Ϫ mice (P Ͻ 0.001). The higher induction of the type I interferon response in the BMM derived from the CLEC5A mice was associated with upregulated TLR3 mRNA after influenza virus infection or PIC treatment (Fig. 6C). Overall, BMM derived from CLEC5A Ϫ/Ϫ mice showed reduced TNF-␣ and IP-10 but increased IFN-␣ compared to the WT mice after infection with the VN HA,NA virus or treatment with PIC. The increased IFN-␣ after influenza infection could be related to the upregulation of TLR3 mRNA in CLEC5A Ϫ/Ϫ  mice, as observed after PIC treatment. The differential type I IFN response in human and murine macrophages suggests that there are different levels of cross talk between CLEC5A-mediated signaling and other cases of PRR-mediated signaling in these two species.
CLEC5A ؊/؊ mice with reduced inflammatory responses but lung viral loads comparable to those of the WT mice were only moderately protected after lethal influenza challenge. We further determined the impact of CLEC5A on influenza pathogenesis in CLEC5A Ϫ/Ϫ and WT mice after lethal challenge with 5 50% murine lethal doses (MLD 50 ) (4,766 PFU/mice) of the VN HA,NA virus. On days 1, 4, and 7, mouse lungs were collected to determine the viral titers and levels of cytokines and chemokines. Lower levels of TNF-␣, IFN-␤, IFN-␥, IP-10, MIP-1␣, and MCP-1 were observed in lung homogenates of CLEC5A Ϫ/Ϫ mice on day 7 postinfection (Fig. 7A) in spite of comparable lung titers in the CLEC5A Ϫ/Ϫ and WT mice on days 1, 4, and 7 postinfection (Fig. 7B). Corroborated with the proinflammatory cytokine and chemokine levels in lung homogenates, significantly lower numbers of cells were noted in the bronchoalveolar lavage (BAL) fluid collected from the CLEC5A Ϫ/Ϫ mice compared to the WT mice on day 7 postinfection (Fig. 7C) but not on day 3 postinfection. Specifically, significantly lower numbers of B cells, CD8 ϩ T cells, ␥␦ T cells, and NK cells were noted in the CLEC5A Ϫ/Ϫ mouse lungs (Fig. 7D). The reduced number of CD8 ϩ T cells and NK cells correlated with the lower IFN-␥ concentration detected in the CLEC5A Ϫ/Ϫ mouse lungs at day 7  (Fig. 7E) compared to the WT (Fig. 7F) mouse lungs at day 7 postinfection. Despite the differences in lung inflammatory responses on day 7 postinfection, comparable weight loss was observed in both WT and CLEC5A Ϫ/Ϫ mice (Fig. 7G). A marginally higher survival rate was observed in CLEC5A Ϫ/Ϫ mice (4/23, 17.4%) compared to wild-type mice (1/25, 4%) (Fig. 7H) (P ϭ 0.014 by log-rank test). We observed a more prominent cytokine reduction in the mouse lungs (Fig. 7A) than that observed in BMM (Fig. 6B) derived from CLEC5A Ϫ/Ϫ and WT mice after influenza infection, possibly due to the mixed cell types present in the mouse lungs. At a lower dose of inoculum (953.2 PFU/mouse, or 1 MLD 50 /mouse), the difference in survival was more prominent, as 90% (9/10) of the CLEC5A Ϫ/Ϫ mice survived the lethal challenge compared to 40% (4/10) of the matching wild-type mice (Fig. 8A). While there was no difference in mouse weight change (Fig. 8B) or lung viral titers ( Fig. 8C), significantly lower proinflammatory cytokine levels were observed in the CLEC5A Ϫ/Ϫ mouse lungs on day 4 (IP-10, IL-1␤, MCP-1, IFN-␥, MIP-1␣, and MIP-1␤), or day 7 (TNF-␣, IFN-␥, and MIP-1␣) postinfection (Fig. 8D). Compared to the cytokine levels detected in the CLEC5A Ϫ/Ϫ and WT mice after infection with 5 MLD 50 (Fig. 7A), we noted an earlier reduction of the proinflammatory cytokines and chemokines in the CLEC5A Ϫ/Ϫ mice than the WT mice (Fig. 8D). Since CLEC5A is restricted to myeloid cells, reducing the dose of inoculation allowed us to better assess the potential proinflammatory role of CLEC5A, as inoculation at a higher dose may lead to substantial replication and induction of proinflammatory cytokines in the epithelial cells where the virus replicated equally well in both the CLEC5A Ϫ/Ϫ and WT mouse lungs. These results also suggest that early reduction of the proinflammatory response is associated with a better survival outcome (as seen with the low-dose challenge). Overall, the data support that CLEC5A mediates induction of proinflammatory cytokine myeloid cells that may lead to differential immune cell infiltration to the lungs and tissue damage.

DISCUSSION
A series of complex virus-host interactions determine the clinical outcome of influenza infections. Here, we report the identification and validation of CLEC5A as a PRR on myeloid cells that mediates enhanced inflammatory responses after influenza virus infection. In human M-M or GM-M, we demonstrated that knocking down CLEC5A expression by gene silencing, blocking influenza-CLEC5A interactions with anti-CLEC5A antibodies, or dampening CLEC5A-mediated signaling using a Syk inhibitor consistently reduced levels of proinflammatory cytokines without affecting the replication of influenza viruses of different subtypes. In murine BMM, reduced IP-10 but elevated IFN-␣ levels were observed in cells derived from the CLEC5A Ϫ/Ϫ compared to the WT mice after influenza infection. Upon lethal challenges at 5 MLD 50 or 1 MLD 50 , CLEC5A Ϫ/Ϫ mice with reduced inflammatory response and immune cell infiltration in the lungs showed significantly improved survival compared to the WT mice, despite comparable lung viral loads between the CLEC5A Ϫ/Ϫ and WT mice throughout the course of infections. The survival difference between the WT and CLEC5A Ϫ/Ϫ mice was more prominent when challenged at a lower lethal dose. Overall, we demonstrated that dampening the CLEC5A-mediated proinflammatory cytokine response in myeloid cells may lower the excessive inflammatory response and improve survival after influenza infections. The results highlight the Syk-coupled CLEC5A signaling pathway as a potential target for immunomodulators to alleviate proinflammatory response associated with influenza immunopathogenesis.
CLEC5A has been demonstrated to mediate inflammatory responses in human or murine macrophages upon DV or JEV infection (26,37). DV mainly targets monocytes for viral replication, and macrophages are considered a major source of proinflammatory cytokine induction during the course of infection (26). Blocking DV-CLEC5A interaction with intravenous injection of anti-CLEC5A blocking antibodies significantly dampened the serum levels of proinflammatory cytokines in DV2-infected STAT1 Ϫ/Ϫ mice and provided protection against DV2-induced complications, including hemorrhage and plasma leakage, leading to a better survival rate compared to the isotype control antibody-treated mice (26). While influenza can similarly activate CLEC5Amediated inflammatory responses in the myeloid cells, influenza viruses predominantly replicate at the respiratory epithelium cells. In addition, the mechanisms of influenza virus or flavivirus interaction with CLEC5A may differ, since some of the anti-CLEC5A blocking antibodies previously shown to successfully dampen cytokine induction after DV or JEV infection failed to suppress the secretion of IP-10 in M-M after influenza virus infection (Fig. 5B). This suggests that the active binding site of CLEC5A for influenza virus is different from that for DV or JEV. The binding sites for the anti-CLEC5A antibodies capable of suppressing cytokine induction after DV, JEV, and influenza virus infection should be further investigated to understand the nature of the ligands for CLEC5A activation.
The cytokine profiles between human PBMC-derived macrophages and murine BMM after influenza infections do not match fully, which could be due to differences in CLEC5A-mediated signaling or differential cross talk with other signaling pathways in these two species. Interspecies differences in the transcriptional response in human and murine macrophages after TLR4-mediated LPS stimulation have been reported (43); in addition, the inducible nitric oxide synthase (iNOS) gene can be induced by IFN-␥ and LPS stimulation in murine macrophages but not in human macrophages (44). These studies suggest that despite significant evolutionary conservation in the pattern recognition receptors, differences in the downstream gene expression profile between mouse and human can still be observed. We examined available literature aiming to understand the impact of CLEC5A-mediated activation on cytokine production in murine BMMs. Activation of CLEC5A in a murine 32Dcl3 myeloid precursor cell line with agonist antibodies was reported to induce higher mRNA levels of RANTES, IP-10, and CCL22 but not TNF-␣ (45). This is consistent with our observation that the CLEC5A Ϫ/Ϫ BMMs secreted significantly lower levels of IP-10 but not TNF-␣ than the CLEC5A ϩ BMMs after influenza infection. However, we observed a differential IFN-␣ response triggered in human and murine macrophage after influenza infection, which could be due to differential cross talk between CLEC5A-mediated signaling and other signaling pathways in human and murine macrophages after influenza infection. It is also not known if functional compensation occurs in the CLEC5A knockout mice. Regardless of the differential cytokine profiles exhibited in human macrophages and murine BMM after influenza infections, the proinflammatory role of CLEC5A is consistently shown in both species with different experimental results, including the reduction of proinflammatory cytokines in the macrophages or in the mouse lungs. Further studies are needed to investigate CLEC5A-mediated signaling and the potential cross talk with other signaling pathways in these two species.
Multiple PRRs work in synergy to detect viral RNA or proteins synthesized during influenza replication (46)(47)(48). A well-orchestrated host response may help to maintain the inflammatory response while inducing an effective adaptive immune response for viral clearance. We observed survival advantage in CLEC5A Ϫ/Ϫ mice after lethal influenza challenge, while a more prominent survival advantage was observed at a sublethal dose. This is consistent with a report showing that mice deficient in TLR3 showed variable survival compared to WT mice depending on the dose or strain of influenza virus used for challenge (49,50). The more prominent survival advantage for the CLEC5A Ϫ/Ϫ mice over the WT mice at a lower dose of inoculum suggests that in addition to the proinflammatory response elicited in the myeloid cells, viral replication kinetics in the lung epithelium cells and the subsequent release of cytokines from the infected cells also determine the survival outcome. The reduction of proinflammatory cytokine secreted by CLEC5A-expressing monocytes/macrophages recruits lower numbers of NK, B, and T cells to the site of infection, which likely lead to reduced lung damage and eventually increased mouse survival.
Taken together, we identified the Syk-coupled CLEC5A as a PRR for influenza virus that mediates proinflammatory responses in human and murine myeloid cells. Without affecting viral load, dampening CLEC5A-mediated inflammatory responses in myeloid cells provided survival advantage in vivo while reducing NK, B, and T cell infiltration into and tissue damage of the mouse lungs. Our results highlight the Syk-coupled CLEC5A signaling pathway as a potential target for immunomodulators to alleviate proinflammatory responses associated with influenza immunopathogenesis.

MATERIALS AND METHODS
Generation of pseudoparticles expressing influenza HA. Pseudoparticles expressing influenza HA were produced as described previously (51). Briefly, the coding sequences of HA derived from VN1203 (H5N1) (PP-VN HA ) or HK5498 (H1N1) (PP-HK HA ) were cloned into the pcDNA3.1 vector (Invitrogen) with an 8-amino-acid FLAG peptide (DYKDDDDK) added to the HA C terminus. Lentiviral packaging vector plasmid pNL Luc E Ϫ R Ϫ , which encodes an HIV-1 provirus lacking a functional env gene and carries a firefly luciferase reporter gene in place of the nef gene (provided by Pierre Charneau, Institute Pasteur, Paris), were cotransfected with pcDNA3.1 with or without coding influenza HA sequence in 293T human embryonic kidney cells using a CalPhos mammalian transfection kit (Clontech). Neuraminidase (5 mU/ml) derived from Vibrio cholerae (Roche) was added to the medium at 36 h posttransfection. The culture medium was harvested at 72 h posttransfection and was further concentrated by sucrose gradient.
Enzyme-linked immunosorbent assay (ELISA) using soluble human CLR and pseudoparticles expressing influenza HA. Soluble CLRs were produced as described previously (26). CLRs were diluted using dilution buffer (1% polyvinyl alcohol, 2 mM MgCl 2 , 2 mM CaCl 2 in Tris-buffered saline) to the concentration of 5 g/ml and were coated on microtiter plates at 100 l/well (0.5 g per well) to capture PP-VN HA or PP-HK HA . The bound pseudoparticles were detected by anti-H1 (Abcam) or anti-H5 antibodies (a generous gift from Robert Webster, St. Jude Children's Research Hospital) followed by their respective horseradish peroxidase (HRP)-conjugated secondary antibodies with 3,3=,5,5=-tetramethylbenzidine substrate (Invitrogen).
Viruses. Recombinant PR8 expressing HA and NA derived from either VN1203 (VN HA,NA ) or HK5498 (HK HA,NA ) was generated as described previously (52). The VN HA,NA virus was a vaccine seed strain, kindly provided by Robert Webster, with its multibasic HA cleavage site replaced by that of a low-pathogenicity avian influenza virus (53). For UV inactivation, influenza viruses were irradiated with UV light (CL-100 ultra violet cross-linker) for 15 min.
Preparation of human PBMC-derived macrophages and murine bone marrow-derived macrophages. Human PBMC were separated from buffy coats of healthy donors (Hong Kong Red Cross Blood Transfusion Service) using Ficoll-Paque density gradient (GE Healthcare Life Sciences) under Institutional