key: cord-0689812-d35gys6r
authors: White, Mitchell R.; Hsieh, I‐Ni; De Luna, Xavier; Hartshorn, Kevan L.
title: Effects of serum amyloid protein A on influenza A virus replication and viral interactions with neutrophils
date: 2020-11-17
journal: J Leukoc Biol
DOI: 10.1002/jlb.4ab0220-116rr
sha: 85ed8c0126e5ffe48b246cf56d06ac6ce6587277
doc_id: 689812
cord_uid: d35gys6r

Innate immunity is vital for the early control of influenza A virus (IAV) infection. Serum amyloid A (SAA1) is an acute phase reactant produced in the liver and lung that rises dramatically during IAV infection. The potential role of SAA1 in host defense against IAV is unknown. SAA1 has been reported to directly activate neutrophils and to recruit them to the lung during infectious and inflammatory processes. Neutrophils are the most abundant cell recruited to the lung in the early phase of IAV infection. There are different forms and preparations of SAA1 that have found to have different effects on phagocyte responses, through various receptors. In this paper, we test the direct effects of various preparations of serum derived or recombinant SAA on IAV and how it modulates the interactions of IAV with neutrophils. All SAA preparations bound to IAV in vitro but caused minimal hemagglutination inhibition or viral aggregation. The human serum‐derived SAA1 or the complex of SAA1 with HDL did have IAV neutralizing activity in vitro, whereas the recombinant SAA1 preparations did not. We found that different SAA preparations also had markedly different effects on neutrophil functions, with E. coli‐derived SAA1 triggering some responses in neutrophils on its own or in presence of IAV whereas mammalian cell‐derived SAA1 did not. This discrepancy could be explained by the reported contamination of the former preparation with bacterial components. Of interest, however, serum SAA alone, serum SAA complexed with HDL, or HDL alone potentiated some neutrophil responses to IAV. Our results suggest that SAA may play some role in host response to IAV, but further work needs to be done to clarify the role of different variants of SAA alone or complexed with HDL.

The innate immune response is critical in containing influenza A virus (IAV) replication during the first few days of infection (i.e., prior to the generation of an adaptive immune response). The innate immune response to IAV is remarkably complex as recently reviewed. 1 Our laboratory has focused principally on the role of soluble inhibitors in respiratory lining fluids and resident or recruited phagocytes. In the current study we evaluate effects of serum amyloid A (SAA1) protein on influenza replication and its interactions with phagocytes. SAA1 is an acute phase reactant and its levels increase markedly Abbreviations: HA, hemagglutinin; HDL, high density lipoprotein; IAV, influenza A virus; SAA1, serum amyloid A 1; SAAHDL, serum amyloid A in complex with HDL; SAP, serum amyloid P. during IAV infection in humans and other animals (e.g., mice, pigs, and ferrets). [2] [3] [4] [5] It has also been recently shown to be markedly elevated in the serum of patients with COVID-19. 6 The effects of other acute phase reactant proteins on IAV have been studied. Surfactant protein D (SP-D) strongly inhibits strains of IAV containing high mannose sugars on their hemagglutinin (HA), through lectin mediated binding to the HA sugars. Serum amyloid P (SAP) and pentraxin both inhibit IAV through a mechanism that has been termed -inhibition. 7, 8 This mechanism involves the presence of sialic acids on SAP or pentraxin, which act as decoy ligands for the viral HA. Two other innate defense proteins that inhibit IAV by this mechanism include surfactant protein A and H-ficolin. [9] [10] [11] Despite the marked elevation of SAA during influenza infection, and proposals to use it as a biomarker of severe infection, we could find no prior studies directly addressing potential anti-influenza activity. SAA has been shown to bind to hepatitis C virus and inhibit infection by this virus through blocking viral entry. 12, 13 SAA is a highly conserved protein through evolution. Normally, the plasma level of SAA is around 1 µg/ml, but it can be induced up to 1000-fold during acute phase response to infections, suggesting a protective role of SAA against acute infections. SAA1 has also been demonstrated in lung lavage at concentrations up to 100 ng/ml during inflammatory conditions. 14, 15 Infection with IAV has been shown to cause marked elevation of lung SAA1 expression. 15 SAA1 binds to HDL and is therefore considered an apolipoprotein. It is believed that SAA1 redirects cholesterol to damaged cells during acute inflammation and injury states. The common form of SAA1 is a hexamer and it binds to heparin through positively charged patches on its surface. 16 SAA1 has been reported to have varied effects on phagocyte activation through binding to various phagocyte receptors including formyl peptide receptor 2 (FPR2), scavenger receptors, TLR2 and TLR4, and the ATP receptor P2X7 . In many cases SAA1 leads to proinflammatory responses; however, anti-inflammatory effects are also reported. 17 SAA1 has been found to act as a chemoattractant for neutrophils by binding to FPR2, 18 and it can induce proinflammatory cytokine production by neutrophils and macrophages. 14,19-21 SAA1 has been shown to delay neutrophil apoptosis through a mechanism involving the P2 × 7 receptor 22 or FPR2 23 in different studies. The apoptosisdelaying effect of SAA1 can be overridden by lipoxins, which also bind FPR2. 23 IL-8 induction by SAA1 in monocytes was mediated by TLR2. 18, 24 Overall these results suggest that SAA1 is a pleiotropic agent mediating effects through multiple receptors. A recent paper showed that a recombinant form of Apo-SAA1 used in many studies activates TLR2 and inflammatory responses due to bacterial lipoprotein bound to the recombinant SAA1 in this preparation. In contrast, recombinant SAA1 produced in HEK cells lacked this effect. 25 This raises the question whether some of the described proinflammatory effects of SAA are mediated by bacterial lipoproteins bound to SAA and not SAA itself. In fact, it was found that SAA3 knockout mice have a proinflammatory phenotype suggesting that SAA may be a predominantly anti-inflammatory protein. 26 SAA3 knockout mice also had increased mortality and viral loads in response to IAV Infection, 26 suggesting either a direct or indirect role for SAA in host defense against IAV. Given the marked rise in SAA in the lung during IAV infection and the findings with SAA knockout mice we decided to test for antiviral activity of SAA. SAA1 has been demonstrated in lung lavage of patients with COPD and its levels correlate with lung neutrophil recruitment, neutrophil elastase, and IL-8 concentrations. 15 Human phagocytes including neutrophils, monocytes, and macrophages play important roles in host defense against IAV. [27] [28] [29] [30] [31] [32] Neutrophils predominate in the early response to IAV infection. This early neutrophilic response has been shown to contribute to viral control and also to modulation of subsequent adaptive responses in several studies. 32 In contrast, other studies have suggested that in some settings exuberant neutrophil responses could be harmful during severe IAV infection. 33 For these reasons we also tested the effects of SAA1 on IAV and neutrophil responses to IAV. Here again we found some discrepancies between the activities of different SAA preparations. Initial studies were done with a serum SAA preparation and the E.coli-derived recombinant Apo-SAA preparation and were started before the report on bacterial lipoproteins in the latter preparation by Burgess et al. 25 We extended the studies to include serum SAA bound to HDL and the mammalian recombinant SAA1 preparation.

The various innate immune proteins used in this study are outlined

in discontinuous sucrose gradient as previously described. 34 were prepared by reverse genetics and propagated in Madin Darby

Canine Kidney (MDCK) cells as described. 35 

Binding of SAA preparations to IAV was tested by ELISA. ELISA plates were coated with 2 µg/ml IAV or 0.05% gelatin as background blocker 

Hemagglutination inhibition was measured serially diluting SAA1 in round-bottom 96-well plates (Serocluster U-Vinyl plates; Costar, Cam-bridge, MA, USA) using PBS as a diluent and then adding IAV to each well. After this human type O red cells were added. Hemagglutination titers were assessed by counting the number of well in which red cell pellets did not form as described. 36 Hemagglutination titers were also assessed on samples taken from the viral aggregation assays. In this case aliquots of the aggregation assay samples were serially diluted followed by addition of red cells.

MDCK cell monolayers were prepared in 96-well plates and grown until confluent. These layers were then infected with diluted IAV preparations for 45 min at 37 • C in PBS. MDCK cells were tested for presence of IAV infected cells after 18 h of virus addition using a monoclonal antibody directed against the IAV nucleoprotein (EMD Millipore, Burlington, MA) as previously described. 36 

Viral aggregation caused by SAA1 was measured by assessing light absorbance at 350 nM by suspensions of IAV. This was done using a Perkin Elmer Lambda 35 UV/Vis spectrophotometer as described. 37 

Neutrophils from healthy volunteers were isolated to >95% purity by using dextran precipitation, followed by Ficoll-Paque gradient separation for the separation of mononuclear cells (layering above the Ficoll-Paque) and neutrophils (below the Ficoll-Paque). The neutrophils were purified further by hypotonic lysis to eliminate any contaminating erythrocytes, as previously described. 34 were pretreated with or without PT (1 µg/ml) for 2 h, or wortmannin (1 µM) for 10 min, or TLR2 antibody (5 µg/ml) for 10 min.

H 2 O 2 production was measured by assessing reduction in scopoletin fluorescence as previously described. 38 In brief neutrophils were added to a mixture of scopoletin, sodium azide, cytochalasin B, and horseradish peroxidase, which were previously shown to maximize detection of IAV induced H 2 O 2 . As previously reported IAV induced respiratory burst occurs at an intracellular location and superoxide anion is not detected, 39 although oxygen consumption and nitroblue tetrazolium assays occur in parallel with H 2 O 2 production. 38 Measurements were made using a POLARstar OPTIMA fluorescent plate reader (BMG Labtech, Durham, NC, USA).

FITC-labeled IAV (Phil82 strain) was prepared and uptake of virus by neutrophils was measured by flow cytometry as described. 40 In brief, IAV was treated with various doses of SAA1 for 30 min at 37 • C. Then it was incubated with cells for 45 min at 37 • C in presence of control buffer. Trypan blue (0.2 mg/ml) was added to these samples to quench extracellular fluorescence. Following washing, the neutrophils were fixed with 1% paraformaldehyde and neutrophil associated fluorescence was measured using flow cytometry. The mean cell fluorescence (2000 cells counted per sample) was measured. 

Statistical comparisons were made using Student's paired, 2-tailed ttest or ANOVA with post hoc test (Tukey's). ANOVA was used for multiple comparisons to a single control.

We tested binding of the SAA preparations to IAV by ELISA. We ing using SAA1 and found no effect (Fig. 1B) . Note that SAA1 (HEKderived recombinant preparation) showed somewhat less binding than the other two preparations. Because SAA1 has a DDK tag we also tested binding using and anti-DDK antibody and this confirmed binding of SAA1 to IAV (Fig. 1C) . In fact, using this method binding of SAA1 to IAV was comparable to binding of the other preparations. In contrast, HDL alone did not bind to IAV (Fig. 1D) .

We next tested various preparations of SAA for viral neutralizing activity. For these studies we included a human serum-derived purified SAA preparation containing amino acids 1-76 of the mature peptide and without the C-terminal tail. Human serum SAA caused doserelated inhibition of infectivity of the Phil82 and PR-8 strains of IAV in MDCK cells ( Fig. 2A ). Unfortunately we were not able to do additional experiments with this preparation because it was no longer produced by Abcam after we completed these assays. The serum SAAHDL preparation also caused neutralization of several strains of IAV (Fig. 2B) ,

whereas HDL alone did not (Fig. 2C ). For these assays we included two preparations Apo-SAA1 and SAA1 (Fig. 2D) with the Phil82 strain of IAV, which was strongly inhibited by the serum SAA preparations. In contrast to the serum preparations, the recombinant preparations had no viral neutralizing activity for IAV (Fig. 2D) .

We next tested if SAA preparations were able to induce viral aggregation. Using a light transmission assay we found a slight amount of viral aggregation induced by SAA1 but not by the Apo-SAA1 or SAAHDL (or HDL alone) (Fig. 3A) . For comparison we included SP-D, which is strong aggregator of IAV. We measured HA titers on the virus samples treated with SAAs used in the aggregation assays.

SAA1 caused some slight aggregation and reduction of HA titer on the treated samples but Apo-SAA1, SAAHDL and HDL did not (Fig. 3B ).

IAV alone caused H 2 O 2 generation by neutrophils (Fig. 4 ) as previously reported. 38 Apo-SAA1 caused dose-related increases in these responses (Fig. 4A ). SAA1 did not cause any similar increase (Fig. 4B ).

at the highest concentration tested (10 µg/ml) (Fig. 4C) . Unexpectedly, HDL alone caused a significant increase in the H 2 O 2 response to IAV.

The SAA and/or HDL preparations (apart from the Abcam serum SAA1, which was not available to test) did not induce neutrophil H 2 O 2 generation on their own (i.e., in absence of IAV; data not shown; n = 3). We also tested if pre-incubation of IAV with SAAs increased the ability of neutrophils to take up the virus (Fig. 5) . The only SAA preparation to significantly increase virus uptake was Apo-SAA1. SAA1, SAAHDL, and HDL did not increase viral uptake.

Neutrophil intracellular calcium release is a precursor to neutrophil activation for many membrane-acting stimuli. As shown in In contrast, no calcium response was elicited by SAA1. IAV alone triggered intracellular calcium release as previously reported. 34 The peak IAV induced response was increased by pre-incubating the virus with Apo-SAA1, SAAHDL, or HDL but not by SAA1 (Fig. 7) . The Apo-SAA1 also stimulated neutrophil IL-8 (CXCL8) production in the absence of IAV (Fig. 8A) , whereas SAAHDL did not and SAA1 caused a minimal response only at the highest concentration tested. For these experiments an OD of 1 was equal to 800 pg/ml and an OD of 0.65 was equal to 400 pg/ml of IL-8. We also tested the effects of lower concentrations of Apo-SAA1 alone or in combination with IAV (Fig. 8B) . As previously reported IAV alone caused robust production of IL-8. Apo-SAA1 had additive effect with IAV at the highest concentration of Apo-SAA1 tested in this assay. We assessed the role of TLR2 receptors or other signaling mechanisms in the neutrophil responses triggered by Apo-SAA1 as shown in Table 2 .

Apo-SAA1 again appeared to increase neutrophil H 2 O 2 responses to IAV and neutrophil uptake of IAV and these effects were partially or fully blocked by anti-TLR2 antibodies (significant for uptake assay). PT and wortmannin inhibited direct calcium responses of neutrophils to Apo-SAA1.

To test the effects of SAA on neutrophil apoptosis we measured neutrophil caspase 3 activity. IAV alone caused acceleration of neutrophil apoptosis as previously described 43 (Fig. 8C ). Apo-SAA and SAAHDL did not alter caspase 3 activation caused by IAV. However, when IAV was pre-incubated with SAA1 or HDL the virus no longer caused a significant increase in caspase 3 activation as compared to control media.

We show for the first time that various preparations of SAA bind to IAV.

Binding was consistently found for the three SAA preparations neutrophils. 45 However, we confirmed neutralizing activity found with serum SAA and SAAHDL against several other strains as well (including a pandemic strain), because this was a novel finding. Note that again the HDL component in SAAHDL could not account for antiviral activity because HDL alone actually increased viral infectivity in this assay. The mechanism through which HDL increased viral infectivity is not clear at this point and requires further study, but note that it also increased neutrophil H 2 O 2 and calcium responses to the virus. HDL has been reported to reduce some proinflammatory effects of SAA (e.g., activation of inflammasomes) 46 so it is of interest that it did not block SAAs viral neutralizing activity in this study.

We cannot at present explain the differences in direct antiviral activity of the serum and recombinant preparations, although prior studies of proinflammatory activities have found some differences between serum-derived SAA1 and recombinant SAA1. 17, 47 The various SAA preparations differ in length (i.e., the serum SAA preparation obtained from Abcam is 76 amino acids and lacks the C-terminus) and the two recombinant preparations are of different lengths (103 for Apo-SAA1 and 122 for SAA1). The HEK cell-derived SAA1 contains the signal peptide, which would be cleaved in vivo. These features could possibly have affected observed antiviral activity. In any case the observed antiviral activity of the serum preparations suggests a possible role of SAA in limiting viral replication, which may in part account for the finding of more severe IAV infection in SAA3 knockout mice. 26 As noted in the introduction, there have been divergent findings regarding the ability of SAA to activate phagocytes. 17 Many recent studies have used the Apo-SAA1 recombinant preparation because it has minimal endotoxin contamination. However, the recent study by Burgess et al. showed that this preparation is contaminated with other bacterial products. 25 We began our studies with Apo-SAA1 prior to that report and basically confirmed and extended on their findings.

We found that the Apo-SAA1 had numerous neutrophil activating effects either alone or in combination with IAV. On its own it induced neutrophil intracellular calcium release and IL-8 production (the latter was also reported by Burgess et al.) . 25 Apo-SAA1 also increased neutrophil respiratory burst and calcium responses to IAV and increased neutrophil uptake of the virus. In contrast, none of these effects were seen with HEK cell-derived SAA1. We think the most likely reason for these differences is the presence of bacterial lipoprotein products in Apo-SAA1, likely due to preparation in E. coli. Experiments with antibodies to TLR2 and metabolic inhibitors were consistent with this interpretation. Note that the HEK cell-derived preparation contains the signal peptide of the protein, which could have impacted on results and future studies using mammalian recombinant or serum-derived SAA1 without the signal protein will be important to fully understand its biologic activities. In addition, we did note variation in neutrophil responses among our assays and also variation in neutrophil stimulating effects of different virus stocks used. These variations are commonly observed even with highly consistent methodology but should be taken into account when comparing results of neutrophil respiratory burst assays especially. Nonetheless, Apo-SAA1 differed from the other preparations in a range of neutrophil assays and the results remain consistent with presence of bacterial products in this preparation. Because SAA1 is known to bind bacterial products, rigorous studies of its effects with and without such products will be of interest (e.g., the effects of SAA1 combined to bacteria or viruses may also be relevant in vivo).

The situation becomes more complicated when we evaluate SAAHDL, which did cause increases in neutrophil intracellular calcium response alone or with IAV and also slight increases in respiratory burst response to IAV. However, in this case these responses were also found with HDL alone. In fact, the responses were more obvious with HDL alone. It may be, therefore, that the neutrophil effects of SAAHDL SAA has been reported to inhibit apoptosis of various cells including neutrophils 22, 23 and dendritic cells. 49 We have previously reported that IAV accelerates apoptosis of neutrophils 43, 50 and that this effect is linked to respiratory burst activation. We here show that SAA1 and HDL were able to blunt the caspase 3 activating effect of IAV, whereas

Apo-SAA1 and SAAHDL were not. In the case of Apo-SAA1 as compared to SAA1 (HEK cell derived), this might reflect increased activation of the cells by the former preparation. Further research on effects of SAA1 (not associated with bacterial products) on neutrophil apoptosis will be of interest.

As noted in the introduction, serum SAA rises markedly during IAV infection and expression in the lung is also increased in this context. Figure 6 . In these experiences, IAV alone or IAV pre-incubated with the indicated concentrations of SAA and or HDL preparations were added to neutrophils. Instances where the IAV induced calcium response was significantly further increased by SAA and or HDL are indicated by * symbols in the legend. * = P < 0.05 and ** = P < 0.01 compared to virus alone. Results represent mean ± SEMfor 3 to 5 experiments F I G U R E 8 Effect of SAA and or HDL on neutrophil IL-8 production or caspase activity-In panel A, IL-8 production by neutrophils was measured by ELISA as described in Section 2 ("Materials And Methods"). ** = P < 0.01 compared to control. Results represent mean ± SEMfor 3 to 5 experiments. Panel B shows IL-8 production in response to influenza A virus (IAV) alone or IAV combined with Aposerum amyloid A (Apo-SAA1). Panel C shows caspase 3 activation by IAV alone or IAV combined with Apo-SAA1, SAAHDL (SAA complexed with HDL), SAA1, or HDL. * = P < 0.05 compared to results with neutrophils in media alone. Wortmannin 98 ± 2 ** Assays were performed as described in Section 2 ("Materials And Methods"). * = P < 0.05 and ** = P < 0.01 compared to control and results represent mean ± SEM for 4 to 5 experiments with separate neutrophil donors. 

The amazing innate immune response to influenza A virus infection

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C-reactive protein, haptoglobin, serum amyloid A and pig major acute phase protein response in pigs simultaneously infected with H1N1 swine influenza virus and Pasteurella multocida

Response of C-reactive protein and serum amyloid A to influenza A infection in older adults

Acute phase response of serum amyloid A protein and C reactive protein to the common cold and influenza

Clinical characteristics of 140 patients infected with SARS-CoV-2 in Wuhan

Serum amyloid P is a sialylated glycoprotein inhibitor of influenza A viruses

A single amino acid substitution in the hemagglutinin of H3N2 subtype influenza A viruses is associated with resistance to the long pentraxin PTX3 and enhanced virulence in mice

Surfactant protein A genetic variants associate with severe respiratory insufficiency in pandemic influenza A virus infection

Surfactant protein-A-deficient mice display an exaggerated early inflammatory response to a betaresistant strain of influenza A virus

Human H-ficolin inhibits replication of seasonal and pandemic influenza A viruses

Human serum amyloid A protein inhibits hepatitis C virus entry into cells

Serum amyloid A has antiviral activity against hepatitis C virus by inhibiting virus entry in a cell culture system

Serum amyloid A promotes lung neutrophilia by increasing IL-17A levels in the mucosa and gammadelta T cells

Serum amyloid A opposes lipoxin A(4) to mediate glucocorticoid refractory lung inflammation in chronic obstructive pulmonary disease

Serum amyloid A1: structure, function and gene polymorphism

Emerging functions of serum amyloid A in inflammation

Serum amyloid A1alpha induces paracrine IL-8/CXCL8 via TLR2 and directly synergizes with this chemokine via CXCR2 and formyl peptide receptor 2 to recruit neutrophils

Serum amyloid A induces NLRP-3-mediated IL-1beta secretion in neutrophils

Serum amyloid A induces interleukin-33 expression through an IRF7-dependent pathway

LL-37 inhibits serum amyloid A-induced IL-8 production in human neutrophils

Serum amyloid A inhibits apoptosis of human neutrophils via a P2X7-sensitive pathway independent of formyl peptide receptor-like 1

Aspirin-triggered lipoxins override the apoptosis-delaying action of serum amyloid A in human neutrophils: a novel mechanism for resolution of inflammation

Hepatic serum amyloid A1 aggravates T cell-mediated hepatitis by inducing chemokines via Toll-like receptor 2 in mice

Bacterial lipoproteins constitute the TLR2-stimulating activity of serum amyloid A

Serum amyloid A3 is required for normal lung development and survival following influenza infection

Critical role of serpinB1 in regulating inflammatory responses in pulmonary influenza infection

CCR2-antagonist prophylaxis reduces pulmonary immune pathology and markedly improves survival during influenza infection

CCR2+ monocytederived dendritic cells and exudate macrophages produce influenzainduced pulmonary immune pathology and mortality

Influenza-infected neutrophils within the infected lungs act as antigen presenting cells for anti-viral CD8(+) T cells

Neutrophils sustain effective CD8(+) T-cell responses in the respiratory tract following influenza infection

The role of neutrophils during mild and severe influenza virus infections of mice

CXCL10-CXCR3 enhances the development of neutrophil-mediated fulminant lung injury of viral and nonviral origin

Effects of influenza A virus on human neutrophil calcium metabolism

The ability of pandemic influenza virus hemagglutinins to induce lower respiratory pathology is associated with decreased surfactant protein D binding

Evidence for a protective role of pulmonary surfactant protein D (SP-D) against influenza A viruses

Hapivirins and diprovirins: novel theta-defensin analogs with potent activity against influenza A virus

Characterization of influenza A virus activation of the human neutrophil

Human neutrophil respiratory burst response to influenza A virus occurs at an intracellular location

Mechanisms of anti-influenza activity of surfactant proteins A and D: comparison with serum collectins

Antiviral activity of the human cathelicidin, LL-37, and derived peptides on seasonal and pandemic influenza A viruses

Mutations flanking the carbohydrate binding site of surfactant protein D confer antiviral activity for pandemic influenza A viruses

Influenza A virus accelerates neutrophil apoptosis and markedly potentiates apoptotic effects of bacteria

Integrated omics and computational glycobiology reveal structural basis for influenza A virus glycan microheterogeneity and host interactions

Human neutrophil defensins increase neutrophil uptake of influenza A virus and bacteria and modify virus-induced respiratory burst responses

High-density lipoprotein inhibits serum amyloid A-mediated reactive oxygen species generation and NLRP3 inflammasome activation

Endogenous acute phase serum amyloid A lacks pro-inflammatory activity, contrasting the two recombinant variants that activate human neutrophils through different receptors

Suppression of lipopolysaccharideinduced inflammatory response by fragments from serum amyloid A

Serum amyloid A inhibits dendritic cell apoptosis to induce glucocorticoid resistance in CD4(+) T cells

Influenza A virus markedly potentiates neutrophil apoptosis induced by bacteria: role of respiratory burst

This work was supported by NIH ROI HL06981.

The authors declare no conflicts of interest.

https://orcid.org/0000-0002-7196-7433