Biochemical and Biophysical Research Communications 371 (2008) 484–489
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Higher in vitro susceptibility of human T cells to H5N1 than H1N1 inﬂuenza viruses q Yong-Gang Li a,b,1, Pranee Thawatsupha c,1, Malinee Chittaganpitch c, Kamonthip Rungrojcharoenkit a, Gui-Mei Li b, Takaaki Nakaya d, Wattana Auwanit c, Kazuyoshi Ikuta a,b,*, Pathom Sawanpanyalert c a
Section of Viral Infections, Thailand-Japan Research Collaboration Center on Emerging and Re-emerging Infections (RCC-ERI), Nonthaburi 11000, Thailand Department of Virology, Research Institute for Microbial Diseases, Osaka University, Yamada-oka 3-1, Suita City, Osaka 565-0871, Japan c National Institute of Health, Department of Medical Sciences, Nonthaburi 11000, Thailand d International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan b
a r t i c l e
i n f o
Article history: Received 17 April 2008 Available online 1 May 2008
Keywords: Inﬂuenza A virus H1N1 H5N1 Human lymphocyte CD4+ T cell Lymphopenia
a b s t r a c t Patients infected with H5N1 inﬂuenza A virus, who had a severe or fatal outcome, exhibited several characteristic clinical manifestations including lymphopenia. In this study, human CD4+ T-cell lines and healthy donor-derived peripheral blood mononuclear cells (PBMCs) were examined for susceptibility to infection with Thai isolates of H5N1 in comparison to those of H1N1. Although cellular levels were variable between H5N1 and H1N1 in T-cell lines and PBMCs, rates of production of progeny virions were signiﬁcantly higher in H5N1 infections, suggesting a more efﬁcient release of virions. In addition, cytopathogenicity in PBMCs, leading to a decline in CD4+ T-cell numbers, were much severer with H5N1 than H1N1. Thus, human T cells could be an important target for infection with H5N1. Ó 2008 Elsevier Inc. All rights reserved.
Inﬂuenza virus inducing an acute viral disease in the respiratory tract is a major public health concern worldwide. In the last century, three serotypes (H1N1, H2N2, and H3N2) have been adapted to humans to produce pandemic strains. These were generated by a presumed reassortment of the HA and NA gene segments between avian and human inﬂuenza A viruses through the infection of a common host, pigs . In 1997, the highly pathogenic avian inﬂuenza A virus H5N1 crossed the species barrier and caused 18 conﬁrmed human infections in Hong Kong with a case-fatality rate of 33%. Since then, many cases of infection have been reported in several countries. The clinical manifestations of infection include lymphopenia and severe pneumonia progressing to the syndromes of acute respiratory distress and multiple organ dysfunctions [2–6]. A high viral load and subsequent intense inﬂammatory responses [5,7–9] are
q RCC-ERI was established by Research Institute for Microbial Diseases, Osaka University and Department of Medical Sciences, Ministry of Public Health, Thailand. * Corresponding author. Address: Department of Virology, Research Institute for Microbial Diseases, Osaka University, Yamada-oka 3-1, Suita City, Osaka 565-0871, Japan. Fax: +81 6 6879 8310. E-mail address: [email protected]
(K. Ikuta). 1 These authors contributed equally to this work.
0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.04.123
thought to be central to the pathogenicity of H5N1 in humans. However, there is no apparent evidence that H5N1 exerts its lethal effects by inducing a cytokine storm. A recent report addressed this issue by demonstrating that genetic deﬁciencies in or chemical suppression of inﬂammatory cytokines did not protect mice against a highly lethal human H5N1 isolate . H5N1 infections in humans are characterized by high pharyngeal viral loads as well as the frequent detection of viral RNA in the rectum and blood. Interestingly, viral RNA in blood was present only in fatal H5N1 cases and was associated with high pharyngeal viral loads . Tsuruoka et al. reported the detection of the genomic viral RNA by reverse transcriptase (RT)-polymerase chain reaction (PCR) in peripheral blood mononuclear cells (PBMCs) from 3 of 18 infected children during an outbreak of H3N2, the sequence of which was identical to that in throat swab ﬂuid . Several immune cells, such as dendritic cells, primary monocytes/macrophages, and T cells, have been shown to be susceptible to H1N1 and H7N7 [12,13]. Recently, human dendritic cells were reported to be susceptible to H5N1 . However, to our knowledge there is no report on the in vitro susceptibility of human lymphocytes to H5N1. In this study, human CD4+ T-cell lines and PBMCs were comparatively examined for possible susceptibility to H1N1 and H5N1. Our results revealed their susceptibility to both viruses. Notably,
Y.-G. Li et al. / Biochemical and Biophysical Research Communications 371 (2008) 484–489
the H5N1 virus recovered from infected PBMCs was much more infectious than the H1N1 virus. Furthermore, H5N1 exhibited a severer cytopathogenicity than H1N1 in the PBMCs. Materials and methods Viruses and cells. Three isolates of each of H1N1 and H5N1, from patients in Thailand were used: A/TH/336/06, A/TH/344/06, and A/ TH/37/05 of H1N1 and A/Kan/353/04, A/PCB/2031/04, and A/SP/83/ 04 of H5N1. All were isolated from swab ﬂuids through propagation in Madin-Darby canine kidney (MDCK) cells . The inocula used were prepared by injecting 11-day-old embryonated chicken eggs. MDCK cells were passaged in Dulbecco’s modiﬁed minimal essential medium (DMEM) containing 10% fetal calf serum (FCS). Human CD4+ T-cell lines (MOLT-4, Jurkat, MT-2, and MT-4) were maintained in RPMI-1640 medium containing 10% FCS. PBMCs were prepared by centrifugation from a single healthy donor or pooled blood from three healthy donors through Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden). The PBMCs were stimulated with
phytohemagglutinin (PHA: 5 lg/ml) for 3 days before being infected, since the susceptibility of PBMCs to inﬂuenza virus was shown to be increased after stimulation with PHA . PBMCs were cultured in RPMI-1640 medium containing 10% FCS. Plaque assay. To titrate the infectivity, a plaque assay was performed . Brieﬂy, MDCK cells at 6 105/well were plated in 6well microplates one day before the assay. The conﬂuent cells were incubated for 1 h at 37 °C with serial 10-fold dilutions of virus sample, then washed with phosphate-buffered saline (PBS) and covered with 1% agarose gel in 2 MEM medium containing 5 lg/ml of TPCK-trypsin (SIGMA). After incubation for 3 days at 37 °C, the agarose gel was removed and the cells were ﬁxed with 10% formaldehyde, then stained with 0.1% crystal violet to visualize the plaques. The infectivity titer was expressed in plaque-forming units (PFU). Infection of human T-cell lines and healthy donor-derived PBMCs. The T-cell lines were cultured at 5 105/ml in 60-mm culture dishes for 2 days, then were mock-infected or infected at a multiplicity of infection (MOI) of 1. After incubation for 1 h at 37 °C, with
Fig. 1. Viral protein expression in human CD4+ T-cell lines infected with inﬂuenza A viruses. (A) Human CD4+ T-cell lines, MOLT-4, Jurkat, MT-2, and MT-4, were mockinfected or similarly infected with H1N1 (TH/344/06) and H5N1 (Kan/353/04) at an MOI of 1. After culture for 24 h, the cells were subjected to IF with anti-NP antibody C43. (B) The same mock-infected (M) or infected (I) cell samples used for (A) were subjected to Western blotting with C43. (C) MT-4 cells were mock-infected (M) or similarly infected with three isolates of H1N1, TH/366/06, TH/344/06, and TH/37/05, and of H5N1, Kan/353/04, PCB/2031/04, and SP/83/04. The infected cells were harvested at different time points from 0 to 48 h post-infection, then subjected to Western blotting with C43.
Y.-G. Li et al. / Biochemical and Biophysical Research Communications 371 (2008) 484–489
Fig. 2. Viral protein expression in healthy donor-derived PBMCs infected with H1N1 and H5N1. (A) PBMCs prepared from pooled blood of three donors were mock-infected (M) and infected with three isolates of H1N1 (TH/366/06, TH/344/06, and TH/37/05) and H5N1 (Kan/353/04, PCB/2031/04, and SP/83/04) at an MOI of 1. After culture for 8, 16, and 24 h, the cells were subjected to Western blotting with C43. (B) PBMCs prepared were similarly infected as above. After culture for 0, 6, 8, 12, 16, 24, 36, and 48 h, the cells were similarly analyzed by Western blotting.
shaking every 15 min, the cells were washed with PBS and cultured in RPMI-1640 medium containing 2 lg/ml TPCK-trypsin and 0.2% bovine serum albumin (BSA) for different time points. Similarly, PBMCs in 6-well microplates were mock-infected and infected with inﬂuenza A virus at an MOI of 1. After adsorption for 1 hr at 37 °C, the cells were washed with PBS and cultured at 5 105 cells/ml in RPMI-1640 medium containing 2 lg/ml TPCKtrypsin and 0.2% BSA for different time points. Immunoﬂuorescence (IF). The mock-infected and infected cells were ﬁxed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The ﬁxed cells were incubated for 1 h at room temperature with a monoclonal antibody (C43) that similarly reactive with H1N1 and H5N1 nucleoprotein (NP), kindly provided by Dr. Yoshinobu Okuno (The Research Foundation for Microbial Diseases of Osaka University, Kagawa, Japan), followed by FITC-conjugated anti-mouse IgG (Invitrogen). Western blot analysis. The mock-infected and infected cells in 6-well microplates were solubilized in SDS–polyacrylamide gel electrophoresis (PAGE) sample buffer (100 ll per well), then 20 ll aliquots of the samples were applied to 12% gels. After their transfer onto PVDF membranes (Amersham), viral proteins were immunostained with C43 for 1 h at room temperature, followed by horseradish peroxidase-conjugated anti-mouse IgG (Invitrogen). The speciﬁc reactions on the membranes were detected with an ECL Western blotting kit (Amersham Pharmacia Biotech). Real-time PCR. The viral RNA was extracted from the culture ﬂuids of infected cells after culture for 24 h using a QIAamp Viral RNA Kit (QIAGEN), then reverse-transcribed to cDNA using a random primer (SuperScript III First-Strand Synthesis System, Invitrogen). The cDNA (5 ll) was used for real-time PCR with primers for the M gene: 5-ACAAAGCGTCTACGCTGCAG and 5-TTCTAACCGA GGTCGAAACG . The reaction conditions using SYBR Green (ABI)  were modiﬁed slightly to 95 °C for 10 min, then 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Flow cytometry. The PBMCs were mock-infected and infected with H5N1, as above. After incubation for 24 h, the cells were subjected to ﬂow cytometry with anti-CD4 (CD4-APC) and anti-CD8 (CD8-APC) murine monoclonal antibodies (BD Bioscience). Detection of inﬂammatory cytokines. Production rates of the cytokines IFN-a, TNF-a, IL-1b, IP-10, and IL-6 in culture ﬂuids from in-
Fig. 3. Higher release of infectious virions into the culture ﬂuids from healthy donor-derived PBMCs infected with H5N1 than H1N1. (A) PBMCs independently prepared from blood of three donors were infected with three isolates of H1N1 (TH/ 366/06, TH/344/06, and TH/37/05) and H5N1 (Kan/353/04, PCB/2031/04, and SP/83/ 04) at an MOI of 1. After culture for 24 h, the culture ﬂuids of infected cells were subjected to real-time PCR using primers at the M gene. The data are shown as viral RNA copies per ml of the original culture ﬂuid. (B) PBMCs prepared from pooled blood of another three donors were similarly infected as above. After culture for 24 h, the infectivity in the ﬂuids of the infected cells was titrated by the plaque assay using MDCK. Viral titers are expressed as log10 PFU/ml.
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fected PBMCs were estimated at 48 h post-infection by using ELISA kits (BIOSOURCE).
Results Susceptibility of CD4+ T-cell lines to the inﬂuenza A viruses H5N1 and H1N1 Three isolates of each of H1N1 (TH/336/06, TH/344/06, and TH/37/05) and H5N1 (Kan/353/04, PCB/2031/04, and SP/83/04) from patients in Thailand were comparatively examined. First, several human CD4+ T-cell lines were investigated for the susceptibility to infection with TH/344/06 and Kan/353/04. The cells were infected at an MOI of 1 and cultured for 24 h. As shown in Fig. 1A, IF revealed that most of the infected MOLT4, Jurkat, MT-2, and MT-4 cells were stained with the anti-NP antibody. The intensity of the ﬂuorescence in H5N1-infected cells was much stronger than that in H1N1-infected cells. These results were conﬁrmed by Western blotting with the same antiNP antibodies (Fig. 1B). The kinetics of viral protein expression in MT-4 cells was also similar for H1N1 and H5N1 by Western blot analysis, although some variation in the expression levels of viral proteins was observed among the isolates for both H5N1 and H1N1 (Fig. 1C). In addition, production rates of infectious viral particles in the culture ﬂuids of infected T-cell lines were signiﬁcantly higher in H5N1 than H1N1 infections according to the plaque assay, i.e., MOLT-4, Jurkat, MT-2, and MT-4 produced infectious viral particles at 1.1 104, 4.0 104, 1.5 105, and 1.4 104 PFU/ml for H1N1, versus 5.5 105, 6.5 105, 4.5 105, and 4.0 105 PFU/ml for H5N1, respectively. Thus, at least in the human CD4+ T-cell lines we examined, although the viral protein expression was essentially compatible between H1N1 and H5N1, the viral particle production rate was signiﬁcantly higher in H5N1 infections.
Greater susceptibility of human PBMCs to H5N1 than H1N1 Two sets of PBMCs independently prepared from three samples of healthy donor-derived blood were stimulated with PHA for 3 days (Fig. 2A and 2B), then infected at an MOI of 1. After adsorption for 1 h, the cells were cultured for 8, 16, and 24 h (Fig. 2A) or 0, 6, 12, 24, 36, and 48 h (Fig. 2B). Western blot analysis with anti-NP antibody revealed that the cells were susceptible to both H5N1 and H1N1, although the results varied slightly among the isolates, especially for H5N1 PCB/2031/04 and SP/83/04, which expressed viral proteins at lower rates than the others. Thus, the viral protein expression rates in the PBMCs were higher for H1N1 than H5N1. Next, viral genome copy numbers and infectivity titers of the viruses released into culture ﬂuids from infected PBMCs were examined (Fig. 3). Real-time PCR showed some difference in the production rate of progeny viral particles from three donor-derived PBMC samples (Fig. 3A). Essentially, H5N1 produced more particles than H1N1 in all three populations of donor-derived PBMCs. In addition, the viral infectivity titer of progeny particles from PBMCs pooled from three donors was also signiﬁcantly higher for H5N1 than H1N1 when examined by plaque assay using MDCK (Fig. 3B). Next, we focused on the difference in cytopathogenicity between H1N1 and H5N1 (Fig. 4). The PBMCs pooled from three donors were similarly infected with an MOI of 1. At 8, 16, and 24 h post-infection, cell counting was performed. The data revealed a signiﬁcant decline of cell numbers in H5N1, compared to H1N1 (Fig. 4A). This result was conﬁrmed in three sets of PBMCs independently prepared from three donors that were infected with the three isolates of each of H1N1 and H5N1 and cultured for 24 h (Fig. 4B). However, we detected no apparent difference in the production levels of inﬂammatory cytokines (IFN-a, TNF-a, IL-1b, IP-10, and IL-6) by PBMCs between H5N1 and H1N1, although the levels were essentially very low and some variation was observed among PBMCs derived from different donors (data not shown). To clarify the target in PBMCs for the reduction of cell numbers after infection with H5N1, we performed ﬂow
Fig. 4. Severer cytopathogenicity in PBMCs after infection with H5N1 than H1N1. (A) PBMCs mock-infected and infected with three isolates of H1N1 (TH/366/06, TH/344/06, and TH/37/05) and H5N1 (Kan/353/04, PCB/2031/04, and SP/83/04), used for Fig. 2A were subjected to cell counting by trypan blue dye exclusion. (B) PBMCs independently prepared from blood of another three donors were similarly infected. After culture for 24 h, the cells were similarly subjected to cell counting. (C) PBMCs prepared from donors 1–3 were independently mock-infected or infected with H5N1 (Kan/353/04) and cultured for 24 h, then subjected to ﬂow cytometry with anti-CD4 and anti-CD8 antibodies. The data are expressed as percentages of CD4+ and CD8+ T cells in H5N1 infection, compared with mock-infection (100%) in individual donors.
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cytometry with anti-CD4 and anti-CD8 antibodies. As shown in Fig. 4C, in the PBMCs from three donors that were mock-infected or infected with Kan/353/04 at an MOI of 1, the CD4+ T-cell numbers were signiﬁcantly reduced (to 50–60% compared to mock-infections), compared with the CD8+ T-cell numbers (to 70–80% compared to mock-infections). Thus, PBMCs were shown to be a good target for the production of progeny H5N1 particles, which lead to a decline in the numbers of CD4+ T cells. Discussion Our examination of human lymphocytes for possible susceptibility to infection with H5N1 and H1N1, revealed a higher rate of cell-free progeny virus production with a severer induction of cytopathogenicity, especially in CD4+ T cells, for H5N1 than H1N1. H5N1 causes a severe and often fatal disease in humans that is characterized by fulminant pneumonia and multi-organ failure [3,4]. Signiﬁcantly, low peripheral blood CD3+ T lymphocyte counts were observed in fatal cases of H5N1 infections and this inversely correlated with pharyngeal viral loads and therefore indicated an association between lymphopenia and the level of viral replication . In addition, the CD4/CD8 ratio was 0.6 in three fatal cases versus 1.1 in three non-fatal cases . In fact, the lymphocyte count and CD4/CD8 ratio was shown to recover in patients who survived . Our examination using the PBMCs from three donors reﬂects the situation in patients infected with H5N1. Consistent with this evidence, Tumpey et al.  found that mice infected with lethal versus non-lethal H5N1 had signiﬁcantly less numbers of both CD4+ and CD8+ circulating T cells. Thus, lymphocytes may have an important role in the pathogenesis of H5N1 infections. Since the H5N1 genome sequence and antigens could be found in neurons of the brain as well as the T lymphocytes of the hilar lymph-node tissue of infected patients , H5N1 may enter the brain and other extrapulmonary organs through the circulation, as SARS is thought to do . High replication efﬁciency, broad tissue tropism, and systemic replication in the lung as well as brain and spleen seem to determine the pathogenicity of H5N1 in mice and ferrets [14,21]. Similarly, viral RNA was detected at high levels not only in nasopharyngeal specimens of H5N1-infected individuals, but also in blood as well as rectal specimens, especially of H5N1-infected individuals with higher pharyngeal viral loads, while there was no viral RNA in blood in cases of non-fatal H5N1 infections or of human inﬂuenza virus infections, suggesting the detection of viral RNA in blood to be a marker for an overall high viral burden . In fact, the successful isolations of H5N1 from the serum of a patient in Vietnam  and the plasma of a patient in Thailand  were reported, indicating viremia in cases of H5N1 infection. Generally, a positive correlation is believed to exist between IL-6 and TNF-a levels and both symptomatic scores and body temperature in inﬂuenza patients . Notably, H5N1 is more potent inducers of proinﬂammatory cytokines and chemokines in primary human respiratory epithelial cells than H1N1 , although it was reported that inﬂammatory cytokines did not affect the pathogenicity in mice . We detected no apparent difference in the production levels of inﬂammatory cytokines (data not shown). In conclusion, human CD4+ T-cell lines and PBMCs are susceptible to infection by human H5N1, however the susceptibility showed a high degree of variation among the isolates. Of note, H5N1 infections in PBMCs essentially produced more cell-free infectious progeny virus and induced severer cytopathogenicity than H1N1 infections.
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