CD33 is downregulated by influenza virus H1N1pdm09 and induces ROS and the TNF-α, IL-1β, and IL-6 cytokines in human mononuclear cells

Abstract

The influenza A virus (IAV) H1N1pdm09 induces exacerbated inflammation, contributing to disease complications. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), favor an inflammatory response that aids viral replication and survival. A pathway by which spontaneous TNF-α production occurs involves either the reduction of Siglec-3 (CD33) levels or the absence of its ligand, sialic acid. Influenza virus uses sialic acid to enter cells by reducing their expression; however, the role of CD33 in IAV H1N1pdm09 stimulation and its relationship with inflammation have not yet been studied. To evaluate the role of CD33 in proinflammatory cytokine production in IAV H1N1pdm09 stimulation, peripheral blood mononuclear cells from healthy subjects were incubated with IAV H1N1pdm09. We observed that the infection caused an increase in the mRNA expression of proinflammatory cytokines such as TNF-α, interleukin (IL)-1β, and IL-6 and a significant reduction in CD33 expression by monocytes at an early stage of infection. Additionally, suppressor of cytokine signaling 3 (SOCS-3) mRNA expression was upregulated at 6 h, and reactive oxygen species (ROS) production increased at 1.5 h. Moreover, a significant reduction in CD33 expression on the cell surface of monocytes from influenza patients or of IAV H1N1pdm09-stimulated monocytes incubated in vitro was observed by flow cytometry. The results suggest that the decrease in CD33 and increase of SOCS-3 expression induced by IAV H1N1pdm09 triggered TNF-α secretion and ROS production, suggesting an additional way to exacerbate inflammation during viral infection.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-021-00663-4.

Introduction

In 2009, a novel H1N1 influenza A virus (IAV) of swine origin spread globally and was declared responsible for the first pandemic of the twenty-first century[1]. IAV H1N1pdm09 now causes seasonal epidemics with approximately 3 to 5 million severe cases and 10–15% deaths worldwide and represents a major social and economic burden[1].

The first step in the IAV H1N1pdm09 infection of host cells is the attachment of virions to cell surface N-acetylneuraminic acid (sialic acid). Sialic acid is the terminal component of oligosaccharide chains expressed by cell surface glycoproteins and glycolipids. It is an essential component of cellular receptors for influenza viruses[2]. Although the interaction between sialic acid and viral hemagglutinin (HA) shows low affinity, the abundance of sialic acid on the surface of mammalian cells nevertheless results in virions binding with elevated avidity to most cells[3].

Once bound to sialic acid, virions can be internalized and trafficked through the endosomal maturation pathway until endosomal acidification triggers a conformational change in viral HA, exposing its fusion peptide and facilitating the fusion of viral and endosomal membranes. Diverse C-type lectin receptors, such as Dectin-2, Mincle, mannose, and CD33, can recognize carbohydrate structures like sialic acids and are involved in pathogen uptake and antigen presentation by immune cells[4].

CD33 or sialic acid-binding immunoglobulin-like lectin 3 (Siglec-3) is a member of the immunoglobulin superfamily. CD33 is involved in host–pathogen recognition, cell–cell interactions, and the subsequent signaling pathways in the immune system[5]. This receptor specifically recognizes glycoconjugates containing sialic acids[6] and has specificity only for sialic acid, with which it forms extensive molecular interactions[7].

The CD33 molecule has an immunoreceptor tyrosine-based inhibitory motif (ITIM) that is implicated in intracellular signaling pathways[5]. This receptor plays a regulatory role, modulating the inflammatory and immune responses. It was reported that the reduction of CD33 molecules on the cell membranes of monocytes is associated with higher levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-8, and IL-12p70, causing exacerbated inflammation[7]. Two mechanisms have been associated with the reduction of CD33 levels. The first is the reduction of sialic acid on the cell surface to prevent cell–cell interactions and the second is oxidation[8],9 These mechanisms activate the proteasomal degradation of CD33 and downregulation of its mRNA, significantly reducing CD33 surface expression [10], 11. This reduction induces immune cell activation and triggers inflammation. In addition, it has been reported that suppressor of cytokine signaling 3 (SOCS-3) is involved in the regulation of inflammatory diseases[12], high expression of SOCS-3 is induced by proinflammatory cytokines[13], and the binding of SOCS-3 and CD33 increases the proteasomal degradation of CD33[10]. IAV infection induces oxidative stress via NADPH oxidase, and the pretreatment with a scavenger ROS decreases the exacerbated inflammation condition[14]. Also, that CD33 is a receptor involved in host–pathogen recognition and is a modulator of the inflammatory and immune response[5].

IAV H1N1pdm09 causes exacerbated inflammation that compromises the integrity of the epithelial cell barrier, leading to respiratory failure. IAV H1N1pdm09 infection of the airway and alveolar epithelial cells promotes immune cell infiltration into the lung; therefore, immune cells such as macrophages, monocytes, and neutrophils are readily exposed to IAV H1N1pdm09 and infection-induced death[15].

Despite the existence of a vaccine against IAV H1N1pdm09, its rapid mutation rate increases the probability of the appearance of new H1N1 strains that are resistant to the immunity conferred by the vaccines available in each season. Therefore, it is important to elucidate the mechanisms associated with the activation of the immune system. This work aimed to determine the CD33 expression, reactive oxygen species (ROS) production, and inflammatory status in monocytes stimulated with the pandemic H1N1pdm09 virus.

Materials and methods

Study participants

Twenty-one patients with influenza and 24 healthy subjects (18–55 years old) were included in the study. Peripheral blood mononuclear cells (PBMCs) from healthy donors were obtained before influenza caused by IAV H1N1pdm09 worldwide in 2009. The patients were diagnosed based on their clinical symptoms, and influenza infection was confirmed via RT-PCR testing of nasopharyngeal swab samples for H1N1 influenza virus. The eligibility criterion for healthy subjects was negative HIV-1 serology, and the exclusion criteria for healthy subjects included pulmonary diseases, asthma, or upper respiratory tract infections, use of immunosuppressive medication, and immunosuppressive diseases. The use of these samples was approved by the Institutional Review Board. Written consent was obtained from all participants, and the Ethics Committee approved this study (B10-09) at the National Institute for Respiratory Diseases (INER) in Mexico City.

PBMC isolation

PBMCs were obtained from 6 mL of heparinized venous whole blood from 16 healthy volunteers via gradient centrifugation over a Ficoll-sodium diatrizoate solution (Lymphoprep, Nycomed Pharma, Oslo, NOR) following standard procedures[16]. The viability of the PBMCs was 99% as assessed by the trypan blue exclusion test. The PBMCs were used for basal CD33 expression on monocytes by flow cytometry or for in vitro infection.

Cell viability

Cell viability was assessed by Fixable Viability Dye eFluor® 660 Cell Stain (eBioscience, California, USA) according to the manufacturer’s instructions. The cell viability is reported as percentage of live CD33 positive cells (Supplementary Fig. 1).

Gene expression analysis by quantitative real-time PCR (qPCR)

10⁶ PBMCs from healthy subjects were either stimulated in vitro with IAV H1N1pdm09 for 6 h or left unstimulated and then lysed for column-based total RNA extraction using the RNeasy Mini Kit (Qiagen, Hilden, DEU) according to the manufacturer’s instructions. Then, cDNA was synthesized from the RNA using the Superscript First-Strand System (Invitrogen, CA, USA) and a thermal cycler (Veriti, Applied Biosystems, CA, USA). qPCR was performed using the TaqMan assay (Applied Biosystems) for human TNF-α (Hs00174128_m1), IL-1β (IHs0174097_m1), IL-6 (Hs00985639_m1), CD33 (Hs00233544_m1), and SOCS-3 (Hs01000485_g1), and ribosomal RNA (rRNA) 18S was used as the housekeeping gene for normalization. Amplification was performed in a StepOne Plus Sequence Detection System (Applied Biosystems) according to standard conditions (2 min at 50 °C, 10 min at 95 °C, and 40 cycles of 30 s at 95 °C and 60 s at 62 °C). Data were analyzed with the 7500 Fast System SDS software version 1.4 (Applied Biosystems), and the results were reported as the fold change in infected cells relative to the unstimulated condition. A relative quantification method (ΔΔCt) was used for the analysis of gene expression.

CD33 expression in monocytes after incubation with IAV H1N1pdm09 in vitro

10⁶ PBMCs from healthy subjects were added to a 24-well, low-adherence culture plate (Corning, NY, USA) and stimulated with IAV H1N1pdm09 (2 × 10⁶ plaque-forming units, multiplicity of infection [MOI] 2), with uninfected PBMCs (medium) included as a negative control. The cells were incubated for 24 h at 37 °C in a 5% CO₂ atmosphere. Then, cells were recovered and stained with anti-human CD33 monoclonal antibody (mAb) conjugated to phycoerythrin (CD33-PE; Becton Dickinson [BD], CA, USA), and the signal was analyzed by flow cytometry.

ROS quantitation by flow cytometry

10⁶ PBMCs from healthy subjects were either incubated with IAV H1N1pdm09 or left unstimulated; ROS production in the cells was then measured 1.5 h and 24 h after incubation. For this assay, 1 μM of a 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (CH₂DCFDA) probe was added for 30 min at room temperature (RT) in the dark. CH₂DCFDA is a non-fluorescent molecule that is chemically reduced and acetylated. It is converted to a fluorescent form when the acetate groups are removed by intracellular esterase and oxidized by the activity of ROS; thus, it is used as an indicator for ROS production within the cell. The cells were stained with anti-human CD33-PE (BD), washed, and immediately measured by flow cytometry, with 10,000 events being acquired for each condition. The measured ROS levels were expressed as the percentage of double-positive CD33 + ROS + cells.

TNF-α quantitation

The supernatants of PBMCs either stimulated with IAV H1N1pdm09 or left unstimulated were harvested after 6 h, 24 h, or 48 h and frozen at − 20 °C until the determination of TNF-α concentrations by enzyme-linked immunosorbent assay (ELISA) as previously described[17]. Absorbance was read at 450 nm on an HT Multi-Mode Microplate Reader (Hycult Biotech, PA, USA). The samples were analyzed in duplicate, and the results are reported as pg/mL.

CD33 expression in monocytes from patients with IAV H1N1pdm09 infection

PBMCs from patients with influenza-associated pneumonia and healthy volunteers were thawed, resuspended in RPMI 1640 supplemented with 2 mM L-glutamine and 10% pooled decomplemented human serum (Valley Biomedicals, VA, USA), and adjusted to 1 × 10⁶ cells/mL. Cells were counted and their viability assessed by the trypan blue exclusion method (> 95%).

10⁶ PBMCs from influenza patients or healthy subjects were stained with saturating amounts of PE-labeled mAb anti-human CD33, clone P67.6 (BD), and incubated for 15 min at RT in the dark. The cells were washed with wash solution (0.01 M PBS, pH 7.2, Lonza, MD, USA), 2% fetal bovine serum (HyClone™, UT, USA), and 0.1% sodium azide (Sigma-Aldrich, MO, USA). Then, the cells were fixed with 1% paraformaldehyde (Sigma-Aldrich) and stored at 4 °C until acquisition. An average of 10,000 events was acquired for each sample using a FACSAria Fusion flow cytometer (BD), and the signals were analyzed using FlowJo software version 10.0 (FlowJo, LLC, OR, USA). The region on the FSC vs. SSC dot plot containing monocytes was selected, and CD33-PE surface expression was analyzed and reported as mean fluorescence intensity (MFI).

Statistical analysis

The data were analyzed by the nonparametric Mann–Whitney test for group comparisons, and the nonparametric Wilcoxon test was used for the analysis of paired samples. All the results are presented as medians and ranks. The statistical significance threshold for p-values was set at ≤ 0.05. All statistical analyses were run with GraphPad Prism version 6.0 h for Mac OS X software.

Results

Patient characteristics

Twenty-four healthy subjects (18–55 years old) and 21 influenza patients were studied. The clinical and demographic data of the IAV H1N1pdm09 patients are shown in Table 1; the average age was 38 years, 48% of patients were male, and 52% did not present with comorbidities. The influenza patients were classified according to their clinical symptoms as severe (n = 10) when they required invasive ventilation and mild (n = 11) when they did not require any invasive procedures. The most severely infected patients presented with bacterial infections. The healthy subjects did not present with any infection or inflammatory pathologies (n = 24).

Table 1.

Clinical and demographical characteristics of influenza patients

ID	Sex/age	Comorbidity or condition	Bacterial infection	Ventilation	Influenza
1	M/25	None	Negative	No	Mild
2	M/42	None	Negative	No	Mild
3	M/29	None	Negative	No	Mild
4	F/31	None	Negative	No	Mild
5	F/50	Hyperglycemia	NA	No	Mild
6	F/47	Systemic arterial hypertension, obesity	Negative	No	Mild
7	F/45	None	Negative	No	Mild
8	F/23	Rheumatic fever	Negative	No	Mild
9	M/40	Obstructive sleep apnea, COPD	Negative	No	Mild
10	F/30	None	Negative	No	Mild
11	F/74	Diabetes, hypertension, COPD	S. pneumoniae	No	Mild
12	F/22	None	NA	Yes	Severe
13	M/19	Hyperactivity	NA	Yes	Severe
14	M/29	None	Negative	Yes	Severe
15	M/40	Arterial hypertension	S. pneumoniae	Yes	Severe
16	F/28	Migraine	P. aeruginosa	Yes	Severe
17	F/70	Peptic acid disease	P. aeruginosa, S. aureus	Yes	Severe
18	M/27	Asthma	Staphylococcus sp. coagulase (-)	Yes	Severe
19	M/48	None	E. coli	Yes	Severe
20	F/25	None	Streptococcus sp.	Yes	Severe
21	M/41	None	P. aeruginosa	Yes	Severe

M male, F female, COPD chronic obstructive pulmonary disease, NA not available

CD33 expression in healthy human cells stimulated with H1N1 influenza virus

We observed that in vitro incubation with IAV H1N1pdm09 significantly decreased CD33 cell surface expression in live monocytes as analyzed by flow cytometry. Uninfected monocytes expressed CD33 at a median MFI = 693 [176–940], while monocytes infected with IAV H1N1pdm09 showed a significantly decreased expression (median MFI = 315 [148–700]; Fig. 1A). Given that incubation with IAV H1N1pdm09 decreased the cell viability after 24-h post-infection, the CD33 expression was assessed in live cells (Supplementary Fig. 1).

Fig. 1.

Effect of IAV H1N1pdm09 on CD33 protein expression and gene expression of CD33 and SOCS-3 in mononuclear cells from healthy subjects after in vitro infection. Mononuclear cells from healthy subjects with or without IAV H1N1pdm09 (MOI 2) were incubated for 24 h at 37 °C in a 5% CO₂ atmosphere. The cells were then stained with mAb anti-human CD33-PE. A Representative histogram of CD33-PE expression on monocytes from healthy subjects uninfected (medium) or stimulated with IAV H1N1pdm09 influenza virus (left). The column bar graph shows CD33 expression in mononuclear cells culture in medium and after IAV H1N1pdm09 stimulation (n = 16) (right). The horizontal line represents the median, Mann–Whitney U test was used to compare the study groups, and the Wilcoxon test was used for paired samples. B The gene expression of CD33 and SOCS-3 was detected at 6 h. The mononuclear cells were lysed, total RNA was extracted by the column method for cDNA synthesis, and the gene expression was analyzed by qPCR. The ΔΔCt method was used for analysis and the results reported as fold change relative to the uninfected condition (n = 7). The horizontal line represents the median, and the Wilcoxon test was used to compare the experimental conditions

Effect of the H1N1 virus on CD33 and SOCS-3 mRNA expression in healthy human mononuclear cells

The expression of CD33 and SOCS-3 mRNA in monocytes stimulated with IAV H1N1pdm09 was assessed to identify the pathways associated with the reduction in CD33 cell surface expression. We observed that CD33 mRNA levels decreased slightly (median = 0.37 [0.06–3.2]), while SOCS-3 expression increased significantly after 6 h of infection in comparison with uninfected cells (4.2 [1–10]; Fig. 1B).

Proinflammatory cytokine expression in mononuclear cells stimulated with influenza virus

Since it has been reported that IAV H1N1pdm09 induces the expression of proinflammatory cytokines in human mononuclear cells[18], TNF-α, IL-1β, and IL-6 mRNA expression was assessed in mononuclear cells incubated with IAV H1N1pdm09. We observed a significant increase in TNF-α, IL-1β, and IL-6 mRNA expression at 6 h of infection (Fig. 2A). The median fold change in expression induced by IAV H1N1pdm09 compared with uninfected cells (medium) from different healthy subjects (n = 7) was 3.8 [1.7–18] for TNF-α, 4.2 [0.96–9.6] for IL-1β, and 27.66 [7.1–247] for IL-16.

Fig. 2.

Effect of the H1N1 virus on proinflammatory cytokine gene expression in mononuclear cells. A Mononuclear cells from healthy subjects either left uninfected or stimulated with IAV H1N1pdm09 (MOI 2) were incubated at 37 °C in a 5% CO₂ atmosphere for 6 h. Then, the cells were lysed, total RNA was extracted by the column method for cDNA synthesis, and TNF-α, IL-1β, and IL-6 gene expression was analyzed by qPCR. The ΔΔCt method was used for analysis and the results reported as fold change relative to the uninfected condition (n = 7), p < 0.05. B The horizontal line represents the median, and the Wilcoxon test was used to compare the experimental conditions. The TNF-α protein was detected in the culture supernatants recovered after 6 h, 24 h, and 48 h, and TNF-α concentration was determined by ELISA and reported as pg/mL by mononuclear cells from healthy donors (n = 16)

In concordance with the increased expression of mRNA, we observed a significantly increased production of TNF-α from 6 to 48 h after infection. The median concentration of TNF-α was 341 pg/mL [150–698] in supernatants from uninfected cells (medium) and 2573 pg/mL [1762–4105] in IAV H1N1pdm09-stimulated cells after 6 h of culture. After 24 h, the median TNF-a concentration was 787 pg/mL [71–2258] in supernatants from unstimulated cells and 1807 pg/mL [509–4313] in IAV H1N1pdm09-stimulated cells. The corresponding median values at 48 h of culture were 517 pg/mL [317–1363] in uninfected cells (medium) and 1622 pg/mL [1121–4000] in IAV H1N1pdm09-stimulated cells (Fig. 2B).

Effect of IAV H1N1pdm09 on ROS production in human monocytes

It is known that Siglecs are prominently expressed on innate immune cells modulating the production of ROS [19], and it has been reported that the pandemic H1N1 virus induces excessive ROS production in human cells, which contributes to lung injury [14], suggesting a possible relationship between CD33 and ROS. Here, we observed that cells stimulated with IAV H1N1pdm09 caused significant ROS production by human monocytes with a lower expression of CD33 molecules (CD33⁺) after 1.5 h of infection (medium, median = 0.65 [0.084–1.07]; IAV H1N1pdm09, median = 39 [14.1–28.3]) and up to 24 h (medium, median = 4 [1.51–11.5]; IAV H1N1pdm09 = 26 [8.4–49.5]; Fig. 3 A and B).

Fig. 3.

Effect of IAV H1N1pdm09 on ROS production. Mononuclear cells from healthy subjects were left uninfected or stimulated with IAV H1N1pdm09 and incubated for 48 h. For ROS detection, the cells were incubated for 1.5 and 24 h, followed by the addition of the CH₂DCFDA probe for 30 min. The cells were stained with mAb anti-human CD33-PE. The analysis strategy was as follows: the monocyte region was defined from the CD33-PE vs. SSC dot plot and the CD33 + /CH₂DCFDA + double-positives were reported as percentages. A Representative flow cytometric analysis plot for medium or IAV H1N1pdm09 conditions. B ROS production at 1.5 h and 24 h after infection (n = 8)

CD33 expression in patients infected with H1N1 influenza virus

To confirm the decrease in cell surface CD33 expression on human monocytes observed upon in vitro IAV H1N1pdm09 stimulation, we evaluated CD33 expression on monocytes from patients with influenza virus infection by flow cytometry.

We observed that CD33 expression was significantly lower in influenza patients (median MFI = 17 [3–152]) than in healthy subjects (median MFI = 111 [32–2137]; Fig. 4). In addition, we evaluated CD33 expression relative to infection severity; however, there was no difference between the mild and severe influenza groups.

Fig. 4.

CD33 expression on monocytes from influenza patients and healthy subjects. Mononuclear cells from healthy subjects and IAV H1N1pdm09 patients were stained with mAb anti-human CD33-PE. A representative histogram of CD33-PE expression from a healthy subject and IAV H1N1pdm09 patient is shown, and the dot plot shows basal CD33 expression in healthy subjects (n = 24) (open circles) and IAV H1N1pdm09 patients (n = 21) (closed circles)

Discussion

Upon viral infection, proinflammatory cytokine, chemokine, and interferon responses are generated, making the immune response a double-edged sword. On the one hand, these responses are required to eliminate viral pathogens; on the other hand, prolonged responses can lead to chronic infection and significant lung damage[20]. Exacerbated inflammation could damage lung tissue, reduce respiratory capacity, and provoke severe disease and even death[21]. Previously, it was shown that the avian IAV H5N1, pandemic H1N1, and H7N9 viruses provoke the early expression of proinflammatory cytokines such as IL-6 and TNF-α in CD14 + monocytes[18]. In accordance with these reports, we show here that the IAV H1N1pdm09 strain also induces IL-6, TNF-α, and IL-1β expression in mononuclear cells.

It has been reported that macrophages are required for IAV infection of human lymphocytes, which raises the possibility that macrophage facilitating the abortive infection of lymphocytes may play a role in the generation of effective immunity to viral antigens[22]; however, it has also been reported that human blood-derived monocytes could be infected with the H3N2 influenza virus[23]. We did not evaluate whether stimulation with IAV produces a productive or abortive infection in PBMC; therefore, we cannot associate the inflammation induction with these processes. We observed a chronic TNF-α production in mononuclear cells stimulated in vitro with IAV H1N1pdm09 up to 48 h. It has previously been reported using a computational model that inflammation control in the host in response to influenza A virus has a positive effect on host survival[24]. Additionally, it has been observed in a mouse model that blocking TNF-α production is associated with a decrease of inflammatory immune responses and IAV replication, favoring the survival of influenza virus-infected mice; thus, the use of commercial TNF-α blockers was suggested for the treatment of influenza[25].

Different TNF-α secretion pathways have been identified during IAV infection, including through the cell necrosis process[26], after neutrophil and macrophage infection, activation through NF-κB translocation, or by lung epithelial cell activation[25]. However, one early and spontaneous mechanism for the induction of TNF-α secretion is through the downregulation of CD33 on the cell surface of monocytes and macrophages [27]. In this report, we observed a lower expression of CD33 in monocytes from patients infected with the IAV H1N1pdm09 strain, and a direct effect of IAV H1N1pdm09 on CD33 downregulation was observed using an in vitro assay. Additionally, TNF-α production was observed after IAV H1N1pdm09 stimulation in the cells of healthy subjects, suggesting a putative role of CD33 in exacerbating inflammation during influenza infection.

Although the mechanisms that regulate CD33 expression are not well known [10], there are two identified pathways by which CD33 levels are decreased, namely protein degradation induced by SOCS-3 or CD33 mRNA repression[11]. We showed a slight decrease in the expression of CD33 mRNA and a significant increase in SOCS-3 expression at 6-h post-infection. A recent study also showed that the pandemic IAV CA04 strain induced SOCS-3 at 3 h after infection in A549 human cells[28]. Possibly, this mechanism contributes significantly to the reduction of CD33 levels on the cell surface in influenza virus infection. Intracellular SOCS-3 binds to phosphorylated CD33, competing with SHP-1/2 for binding to the CD33 ITIM and leading to proteasomal degradation of complexed SOCS-3 and CD33 [10]. These findings suggest that IAV H1N1pdm09 increases SOCS-3 levels and perhaps causes the degradation of the CD33 protein.

Previously, it was demonstrated that CD33 reduction triggers the spontaneous secretion of TNF-α by peripheral monocytes[8,27]. We observed the release of TNF-α by mononuclear cells after IAV H1N1pdm09 stimulation. We also reported previously that spontaneous TNF-α release by CD33 reduction is induced by ROS. In an in vitro model of high glucose concentrations in human monocytes, the cells showed higher ROS production and CD33 reduction. Furthermore, the pretreatment of mononuclear cell with an ROS scavenger (α-tocopherol) decreased ROS production and restored CD33 expression[9]. Additionally, we demonstrated that the oxidant models generated by peroxide hydrogen and iodoacetate in human monocytes show exacerbated ROS production and CD33 reduction and pretreatment with nordihydroguaiaretic acid (ROS scavenger) prevents ROS production and CD33 reduction[10].

Exacerbated ROS production is well-demonstrated during IAV infection and contributes to inflammatory conditions and lung injury[29]. It was reported previously that IAV could be internalized in macrophages within 1 h, causing the overproduction of ROS and provoking an oxidative burst via NADPH oxidase[14], and CD33-related Siglecs were prominently expressed on innate immune cells modulating the production of ROS[19],30. In this work, we also observed that IAV H1N1pdm09 induced ROS generation from 1.5 h to 24 h after infection in monocytes. In this regard, ROS production induced by IAV H1N1pdm09 likely reduced CD33 expression, suggesting an interplay between ROS, CD33, and IAV, and possibly contributing to exacerbated inflammation.

The study has some limitations, such as the number of IAV patients studied and the pathway of reduction of CD33 by IAV H1N1pdm09; nevertheless, we report that IAV H1N1pdm09 reduced CD33 expression in monocytes from patients infected with influenza virus as well as in monocytes stimulated with IAV H1N1pdm09. The viral stimulation in mononuclear cells induced ROS generation and reduced CD33 expression on monocytes, likely contributing to exacerbate TNF-α production through SOCS-3 overexpression and intensifying lung damage in IAV disease. In conclusion, IAV H1N1pdm09 reduces the CD33 expressed on monocytes and triggers the secretion of TNF-α, suggesting an additional way to exacerbate inflammation during viral infection.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

Conacyt,2010-C01-140942,126693