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Association of Profoundly Impaired Immune Competence in H1N1v-Infected Patients with a Severe or Fatal Clinical Course

  1. Chiara Agrati1,
  2. Cristiana Gioia1,
  3. Eleonora Lalle1,
  4. Eleonora Cimini1,
  5. Concetta Castilletti1,
  6. Orlando Armignacco2,
  7. Francesco Nicola Lauria1,
  8. Federica Ferraro1,
  9. Mario Antonini1,
  10. Giuseppe Ippolito1,
  11. Maria Rosaria Capobianchi1 and
  12. Federico Martini1
  1. 1 National Institute for Infectious Diseases “ Lazzaro Spallanzani” Istituto di Ricovero e Cura a Carattere Scientifico, Rome
  2. 2 Belcolle Hospital, Viterbo, Italy
  1. Reprints or correspondence: Chiara Agrati National Institute for Infectious Diseases L. Spallanzani Via Portuense 292 00149 Rome Italy (agrati{at}inmi.it).
 
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Abstract

Background. Pandemic A/H1N1v influenza is characterized by a mild clinical course. However, a small subset of patients develops a rapidly progressive course caused by primary viral pneumonia or secondary bacterial infections that, in many cases, lead to death due to respiratory failure. The aim of the present study was to analyze the involvement of the immune response in the clinical presentation of H1N1v influenza.

Methods. The differentiation and functional capability of T cells from H1N1v-infected patients presenting with either mild disease (n = 22) or severe or fatal disease (n = 6) were compared. Moreover, plasma cytokines and chemokines were quantified

Results. T cells from H1N1v-infected patients presenting with a severe clinical course resulted in impaired effector cell differentiation and failed to respond to mitogenic stimulation. T cell anergy was strictly associated with a severe acute phase of infection, but T cells could be restored in patients able to recover. Of interest, massive expression of CD95 marker was found on anergic T cells, suggesting an apoptosis-related mechanism. Finally, lower plasma levels of interferon-a and monocyte chemoattractant protein-1 were found in patients with a worse clinical course of influenza, suggesting impaired production of these cytokines.

Conclusions. Our results show a strict association between host immune competence and the severity of the clinical course of H1N1v infection. By monitoring host functional response, patients with an enhanced risk of developing influenza-associated severe complications could be identified in a timely manner.

Pandemic influenza A (H1N1v), which first appeared in April 2009, is rapidly spreading throughout the world [1–4]. Several early reports suggested that the clinical course of pandemic influenza A 2009 is generally mild compared with the clinical course of previous pandemics; however, data on the clinical characteristics and the risk of complications are now emerging. Indeed, the majority of persons infected with H1N1v worldwide experience uncomplicated influenza-like illness, experiencing full recovery within one week, even without medical treatment. However, concern is now focused on the clinical course and management of small subsets of patients who rapidly develop very severe progressive viral or bacterial pneumonia. Respiratory failure and refractory shock have been the most common causes of death [5, 6]

The risk of severe or fatal illness is highest in such patient groups as pregnant women, children <2 years of age, and people with chronic lung disease, obesity, and other morbidities [7–12]. However, both young subjects and adults may present with a severe clinical course of H1N1v infection without having any known risk factor [6, 8, 10, 12], and the underlying mechanisms of pathogenesis still are not completely elucidated.

A major issue regarding the ongoing outbreak of swine-origin H1N1v influenza is that the virus may be so antigenically different from seasonal influenza virus that little immune protection exists in the human population. Nevertheless, the ability to respond to different mitogens and strength of the immune response to H1N1v could play a relevant role in determining disease outcome. A serological analysis showed a very limited cross-reactive antibody response both before and after seasonal vaccination, suggesting the absence of a protective antibody coverage in the general population [13]. Some degree of preexisting immunity was found only in elderly adults (ie, adults >60 years of age), probably as a result of remote exposure to a related influenza A (H1N1) virus [13]. On the other hand, the role of T cell immunity to pandemic H1N1v virus is still not defined. T cell immunity may contribute to virus clearance, reduce the severity of the symptoms, and prevent such complications as secondary bacterial infections, which are known to have played a predominant role as a cause of death during the 1918–1919 “ Spanish flu” pandemic [14, 15]. An interesting analysis of preexisting B and T cell immunity to H1N1v was recently reported [16], showing that T cells are the main actors in the cross-immune recognition of H1N1v. Specifically, a detailed comparison of B and T cell epitopes showed that >60% of T cell epitopes are fully conserved between H1N1v and seasonal H1N1 strains, providing a rationale for cross-recognition of the new virus by T cells. On the whole, although T cell responses do not prevent infection, they do contribute to the clearance of infected cells, and such preexisting immunity could at least partially explain a mild course of disease [17–20].

During influenza infection, an effective immune response needs well-regulated integration between innate and adaptive immunity. A rapid and coordinated production of such soluble innate mediators as type 1 interferons and other cytokines and chemokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1, is essential in the first phases of infection [21–23]. Nevertheless, a virus-driven dysregulation of inflammatory factors leading to cytokine hyperproduction, known as a cytokine storm, was described as contributing to the pathogenesis of human H5N1 disease during highly pathogenetic H5N1 infection [24]. Finally, IFN-α is recognized as one of the earliest innate antiviral factors, playing a key role in limiting virus replication at the primary site of the infection and blocking its spread to distant body sites [25–27].

The aim of this study was to analyze the involvement of T cell immunity in the clinical presentation of H1N1v infection. Specifically, the differentiation profile and functional analysis of T cells from H1N1v-infected patients presenting with mild or severe/fatal disease were compared. Moreover, multiparametric analysis of plasma cytokines and chemokines was performed to define their role in the balance between protection and pathogenesis of H1N1v infection.

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Materials and Methods

Patients and biological samples. Twenty-eight H1N1v-infected patients (mean age, 29.9 years) and 10 healthy control subjects (mean age, 33.4 years) were recruited at the National Institute for Infectious Diseases “ Lazzaro Spallanzani.” The study was approved by the ethics committee of the institute, and all enrolled individuals provided written informed consent. The clinical characteristics of the patients are presented in Table 1. Peripheral blood and nasopharyngeal swab specimens were collected during the acute phase of infection and, for selected patients, during the convalescent phase (as defined by clinical resolution and a negative H1N1v polymerase chain reaction [PCR] test result). As shown in Table 2, H1N1v-infected patients were divided into 2 different groups on the basis of clinical severity: those with mild disease (MD; n = 22) and those with severe or fatal disease (SFD; n = 6). Specifically, patients with MD were identified as patients needing home care, whereas patients with SFD needed care in the intensive care unit. All patients (both those with MD and those with SFD) showed a significant hemagglutination inhibition titer (⩾1:80) after 20 days from the time of symptom onset. Plasma samples were cryopreserved for the analysis of cytokines and chemokines, and peripheral blood mononuclear cells were isolated from whole blood by use of Ficoll density gradient centrifugation (Cedarlane Laboratories) and were cryopreserved at −150°C.

H1N1v quantification in nasopharyngeal swab specimens and viral antigen preparations. Nucleic acids were tested using a real-time reverse-transcriptase (RT)-PCR-based method, in accordance with Centers for Disease Control and Prevention guidelines. The influenza A viral load (VL) in the RT-PCR-positive samples was established with the M gene primer/probe set, using an external quantification curve.

A clinical isolate of H1N1v influenza virus (provided by Alberta Azzi, Florence, Italy) was amplified on Madin-Darby canine kidney cells [28, 29].

Phenotypic analysis. Phenotypic analysis of peripheral blood mononuclear cells from H1N1v-infected patients (n = 16) and from healthy control subjects (n = 8) was performed using flow cytometry. Specifically, T cell subsets were analyzed using monoclonal antibodies (BD Biosciences) against CD3, CD8, and CD4; the differentiation profile was analyzed using monoclonal antibodies directed against CD45RA and CD27; and, finally, apoptotic commitment was assessed using monoclonal antibodies against CD95. In brief, thawed peripheral blood mononuclear cells (5 × 105 cells) were stained with a monoclonal antibody cocktail, washed, and acquired on a FACSCanto II (Becton Dickinson) flow cytometer.

T cell functionality. T cell functionality was evaluated by monitoring IFN-γ production after mitogenic stimulation. Specifically, peripheral blood mononuclear cells from healthy control subjects and from H1N1v-infected patients presenting with MD (n = 10) or SFD (n = 6) were stimulated with phytohemagglutinin (PHA; 5 µg/mL [Sigma Aldrich]) or with phorbol12-myristate-13-acetate (PMA; 50 ng/mL [Sigma Aldrich]) plus ionomycin (10 ng/mL; Sigma Aldrich) for 20–24 h, and the responses were evaluated by IFN-γ enzyme-linked immunospot assay (AID Diagnostika). The viability of peripheral blood mononuclear cells was assessed by Trypan blue exclusion.

A polyfunctional analysis was performed by intracellular cytokine staining. Specifically, peripheral blood mononuclear cells from selected H1N1v-infected patients with SFD in the acute phase (n = 4) and convalescent phase (after 20 and 27 days [t20d and t27d, respectively]) (n = 2) of H1N1v infection were cultured with clinical isolates of inactivated pandemic H1N1 virus or with mitogens (PHA or PMA/ionomycin) for 16–18 h in the presence of brefeldin A (10 µg/mL; Sigma Aldrich). Cells were stained with monoclonal antibodies specific for CD3 and CD4 and, after fixation/permeabilization, with monoclonal antibodies against IFN-γ and IL-2, and they were acquired on a FACSCanto II cytometer (BD Biosciences).

Cytokine and chemokine analysis. Cytokines and chemokines were analyzed in plasma samples by use of a multiparametric assay (a cytometric bead array, or CBA) or by enzyme-linked immunosorbent assay. Quantitative analysis of inflammatory cytokines was performed using CBA (BD Bioscience Pharmingen), allowing the simultaneous measurement of 6 different cytokines (IL-1γ, IL-6, IL-8, IL-10, IL-12, and TNF-α) in a single sample. This assay was performed and analyzed in accordance with the instructions of the manufacturer. A quantitative analysis of plasmatic IFN-α and MCP-1 was determined by enzyme-linked immunosorbent assays (for IFN-α, an enzyme-linked immunosorbent assay from PBL Biomedical Laboratories; for MCP-1, an enzyme-linked immunosorbent assay from R&D Systems).

Statistical analysis. The statistical significance of the results was determined using Prism software (version 4; Graph-Pad). Statistical analysis was performed using the nonparametric Mann-Whitney U test, and differences were considered to be significant when P<.05.

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Results

The T cell frequency and differentiation profile in H1N1vinfected patients. The effects of H1N1v infection on the frequency and differentiation profile of T cell subsets were analyzed in 16 H1N1v-infected patients and 8 healthy donors. As shown in Figure 1A, H1N1v infection is associated with a significant decrease in the frequency of circulating CD3 T cells (median [interquartile range {IQR}] for healthy controls vs. H1N1v-infected patients, 73.2% [65.7%–74.2%] vs. 50.2% [33.3%–65.3%], respectively) (P<.05). Of note, T cell depletion specifically affects CD4 T cells (median [IQR] for healthy controls vs. H1N1v-infected patients, 50.0% [46.6%–55.5%] vs. 29.8% [20.5%–39.8%], respectively) (P<.05). Moreover, the differentiation profile of CD3 and CD4 T cells was analyzed by monitoring surface CD45RA and CD27 marker expression. No differences in CD3 differentiation were observed between H1N1v-infected patients and healthy controls (Figure 1B). In contrast, CD4 T cells from H1N1v-infected patients presented a more advanced differentiation profile (Figure 1C), showing a decrease in the frequency of naive CD4 T cells (median [IQR] for healthy controls vs. H1N1-infected patients, 47.6% [39.0%–61.0%] vs. 26.6% [18.9%–35.1%], respectively) (P<.05) and a parallel increase in the frequency of central memory (CM) T cells (median [IQR] for healthy controls vs. H1N1v-infected patients, 32.2% [25.1%–41.8%] vs. 45.4% [35.9%–55.2%], respectively) (P<.05) and effector memory (EM) T cells (median [IQR] for healthy controls vs. H1N1v-infected patients, 13.8% [8.5%–17.2%] vs. 23.9% [18.7%–31.7%], respectively) (P<.05).

Figure 1.
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Figure 1.

Phenotypic and differentiation analysis of T cell subsets in H1N1v-infected patients and in healthy donors (HD). CD3, CD4, and CD8 T cell frequency (A) was compared in 8 HD (white bars)and in 16 H1N1v-infected patients (H1N1v patients) (gray bars). Moreover, analysis of the differentiation profile of CD3 (B) and CD4 (C) T cells was performed using CD27 and CD45RA markers, allowing for definition of naive (CD27+ CD45RA+), central memory (CM; CD27+ CD45RA−), effector memory (EM; CD27− CD45RA−), and terminally differentiated (TEMRA; CD27− CD45RA+) cells. Statistical analysis was performed using the nonparametric Mann-Whitney U test. * P<.05; ** P<.01.

To understand whether CD4 T cell depletion and differentiation can be associated with a different clinical outcome, we divided H1N1v-infected patients into 2 different groups on the basis of symptoms severity: MD (n = 22) and SFD (n = 6). As shown in Table 1, we did not observe any correlation between different clinical outcomes and the H1N1v load in respiratory secretions (median [IQR] for patients with MD and patients with SFD, 392,752 ×104 copies/mL [4565–11,095,000 ×104 copies/mL] vs. 27,404 ×104 copies/mL [16,219–2,633,500 ×104 copies/mL], respectively); although derived from a sample of limited size that could affect statistical power, this finding suggests that factors other than viral replication may be responsible for severe clinical presentation. The analysis of T cell subsets and the differentiation profile did not find evidence of any correlation between clinical presentation and the frequency of CD3, CD4, and CD8 T cells (Figure 2A) on one hand or the overall differentiation of CD3 T cells (Figure 2B)onthe other hand. In contrast, the CD4 T cell differentiation process was significantly different for those with MD (n = 10) and those with SFD (n = 6). Specifically, clinical severity was associated with a higher frequency of CM CD4 T cells (median [IQR] for patients with MD vs. those with SFD, 38.8% [26.5%–47.4%] vs. 52.1% [41.4%–66.4%], respectively) (P<.05) and a lower frequency of EM CD4 T cells (median [IQR] for patients with MD vs. those with SFD, 27.3% [22.9%–31.6%] vs. 16.4% [7.3%–28.2%], respectively) (P<.05) (Figure 2C), suggesting a block in the differentiation process of CD4 T cells at the CM stage. Altogether, these results suggest selective depletion and differentiation impairment of the CD4 T cell subset associated with a more severe H1N1v infection.

Figure 2.
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Figure 2.

Comparison of the differentiation profile in patients with mild disease (MD) and patients with severe or fatal disease (SFD). Circulating CD3, CD4, and CD8 T cell frequency (A) was compared in H1N1v-infected patients presenting with MD (n = 10) (hatched bars) or SFD (n = 6) (gray bars). Moreover, analysis of the differentation profile of CD3 (B) and CD4 (C) T cells was performed using CD27 and CD45RA markers, as previously described. Statistical analysis was performed using the nonparametric Mann-Whitney U test. CM, central memory cells; EM, effector memory cells; TEMRA, terminally differentiated cells. * P<.05; ** P<.01.

Profound functional anergy of T cells from patients with SFD. To verify whether the CD4 T cell differentiation profile observed in patients with SFD was associated with different functional capability, IFN-γ production by T cells after mitogenic stimulation was analyzed by enzyme-linked immunospot assay (Figure 3A and Table 2). Two different mitogenic stimulations were used: PHA, which acts through CD3 triggering, and PMA/ionomycin, which is known to activate T cells through a TCR/CD3-independent mechanism. As shown in Figure 3A, T cells from patients with MD were able to respond to both PHA and PMA/ionomycin stimulation. In contrast, patients with SFD completely lost PHA responsiveness, suggesting a profound and general impairment of immune response (Figure 3B). Nevertheless, detectable IFN-γ production after PMA/ionomycin stimulation was observed in patients with SFD, suggesting that T cell anergy was associated with defective TCR/CD3 signal transduction rather than with decreased cell vitality or other aspecific causes (Figure 3B). The different response capability of CD3 triggering in H1N1v-infected patients was confirmed in 10 patients with MD and in 6 patients with SFD, as shown in Table 2.

Figure 3.
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Figure 3.

Association of profound T cell anergy with disease severity. T cell functionality was assessed by enzyme-linked immunospot assay after phytohemmaglutinin (PHA) or phorbol-12-myristate-13-acetate (PMA)/ionomycin stimulation during the acute phase of infection in patients presenting with mild disease (MD; n = 10) (A) or severe or fatal disease (SFD; n = 6) (B) and in patients who had recovered from SFD (n = 2) (C). In selected patients with SFD, a polyfunctional analysis of interferon (IFN)-γ and interleukin (IL)-2 production by T cells after PHA (D) or H1N1v-specific stimulation (E) was performed by intracellular staining and flow cytometry (D) in the acute phase of infection (n = 4) and after recovery (n = 2) (after 20 days and after 27 days).

Figure 4.
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Figure 4.

Correlation of increased expression of the CD95 molecule on lymphocytes with clinical outcome. A, Cytometric evaluation of CD95 apoptotic marker expression on T cells from a representative patient. B, Statistical comparison between patients with mild disease (MD; n = 10)(hatched bars) and patients with severe or fatal disease (SFD; n = 6)(gray bars). Statistical analysis was performed using the nonparametric Mann-Whitney U test. ** P<.01.

Figure 5.
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Figure 5.

Correlation of impairment of interferon (IFN)-α and monocyte chemoattractant protein (MCP)-1 production with severe clinical presentation. Plasma IFN-α (A) and MCP-1 (B) levels were evaluated by enzymelinked immunosorbent assay. Statistical analysis was performed using the nonparametric Mann-Whitney U test. ** P<.01.

Table 1.
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Table 1.

Characteristics of H1N1-Infected White Patients with Mild (n = 22) or Severe or Fatal (n = 6) Disease

Table 2.
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Table 2.

Strength of the Phytohemagglutinin T Cell Response

To analyze whether T cell anergy observed during the acute phase of H1N1v infection in patients with SFD was transient, T cell functionality was analyzed after recovery in patients who were able to overcome infection after a severe clinical course (Figure 3C and Table 2). Interestingly, the T cell response to PHA was restored in patients who had recovered from SFD, suggesting a strict temporal association between T cell anergy and the acute phase of H1N1v infection. In patients with SFD, we also assessed the polyfunctional profile of T cells by use of multiparametric intracellular staining and flow cytometric analysis. Specifically, we monitored the frequency of IL-2-, IFN-γ-, and IL-2/IFN-γ-producing T cells after PHA stimulation in 4 acute-phase and 2 convalescent-phase samples (at t20d and t27d, respectively) (Figure 3D). During the acute phase, T cells were unable to produce any IFN-γ or IL-2. In contrast, after recovery, production of both IFN-γ and IL-2 by T cells was progressively restored (Figure 3D), suggesting that T cell anergy is transient and can be reverted in patients who are able to recover. Interestingly, the recovery of T cell anergy also included the H1N1v-specific response, as shown in Figure 3D for one representative patient.

T cell anergy in patients with SFD is associated with a massive expression of apoptosis marker. To analyze possible involvement of apoptosis in T cell anergy, CD95 marker expression on T cells from patients with SFD (n = 6) and those with MD (n = 10) was evaluated by flow cytometry. As shown for a representative subject (Figure 4A), T cells from patients with SFD massively express CD95. Moreover, as shown in Figure 4B, CD95 expression was higher on CD3 cells in patients with SFD than on CD3 T cells in patients with MD (median [IQR] in patients with MD vs. patients with SFD, 40.1% [31.3%–52.4%] vs. 69.4% [63.0%–75.1%], respectively) (P<.01) and on CD4 T cells in patients with MD (median [IQR] for patients with MD vs. patients with SFD, 48.0% [38.9%–67.6%] vs. 77.7% [74.4%–79.5%], respectively) (P<.01), suggesting that T cell hyporesponsiveness in patients with SFD could at least partially be due to apoptosis commitment of CD4 T cells.

Circulating levels of cytokines and chemokines. Cytokine and chemokine production during acute infection represents one of the key events in orienting clinical evolution toward protection and/or pathogenesis. To verify possible involvement of soluble factors in the T cell anergy observed in patients with SFD, plasma levels of cytokines and chemokines were evaluated by a multiparametric assay (IL-1β, IL-6, IL-8, IL-10, IL-12, and TNF-α) and by enzyme-linked immunosorbent assays (IFN-γ, IFN-α, and MCP-1). As expected, increased expression of inflammatory cytokines (IL-6 and IL-8) was found in H1N1-vinfected patients, compared with healthy controls (data not shown). Nevertheless, when comparing patients with MD and patients with SFD, no significant differences were observed in terms of IL-1β, IL-6, IL-8, IL-10, and IL-12 (data not shown). Interestingly, patients with SFD were characterized by significantly lower levels of IFN-α (a 40-fold reduction) and MCP1 (a 1.62-fold reduction), suggesting an impaired production of these cytokines in patients with a worse clinical outcome (Figure 5).

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Discussion

The emergence of a new influenza pandemic by H1N1v created considerable alert among health care authorities about the real threat for human health [2, 4]. One of the main issues regarding H1N1v infection is whether deterioration of the human host is the result of the virus per se, the impairment of the immune system, or a massive cytokine storm [24]. Unfortunately, a defined answer is still missing. We observed that the different clinical outcome of H1N1v infection did not correlate with the viral load, suggesting that factors other than viral replication can be responsible for severe clinical presentation. The aim of this study was to correlate the immune response profile with the clinical course of H1N1v infection, defining possible markers of disease severity.

Phenotypic analysis of T cells showed pronounced lymphopenia that mainly affected CD4 T helper cells, as has been reported for other influenza A infections [30–33]. Several mechanisms for this finding may be hypothesized, including migration to the infected tissues, as well as effective cell depletion due to apoptosis induced by the virus or by bystander processes, and/or depression in hematopoiesis [34]. Moreover, CD4 T cells from H1N1v-infected patients present a more advanced differentiation profile than that noted in CD4 T cells from healthy controls, showing a decrease in naive CD4 T cells and a parallel increase in CM and EM compartments. Of note, the ability to generate effector cells resulted impaired in H1N1v-infected patients presenting with a severe clinical course, suggesting a block in the differentiation process. The expansion of effector cells represents a key event in inducing a protective immune response [35], and the ability of viruses to modulate T cell differentiation was observed in other viral infections and correlated with ineffective immune responses [36].

A robust T cell response is well known to play a major role during influenza infections by reducing the severity of the symptoms and preventing such complications as secondary bacterial infections [17–20]. Impairment of the T cell response to mitogenic and bacterial stimulation during H1N1v infection recently has been reported elsewhere [33]. Both general and H1N1v-specific T cell anergy, in terms of IFN-γ and IL-2 production, specifically affects H1N1v-infected patients with SFD, suggesting a strong association between T cell anergy and clinical severity and thus confirming a central role of cell-mediated immunity in reducing the severity of clinical diseases [17–20]. Interestingly, after recovery, patients with SFD had full restoration of their ability to produce both IFN-γ and IL-2, suggesting functional restoration of both effector and memory immune compartments.

The occurrence of severe H1N1v infections as well as complete T cell anergy was observed only in a minority of H1N1vinfected patients. Many possible molecular mechanisms could be responsible for this complete T cell anergy. It is likely that the expansion of T regulatory cells [33] or the induction of T cell apoptosis may be involved in this immunodeficiency induction. Several reports from in vitro or animal models described the ability of influenza viruses to induce apoptosis in monocytes and lymphocytes through Fas/FasL or TRAIL mechanisms [34, 37–40]. In our study, the expression of CD95 on CD4 T cells was observed in vivo in H1N1v-infected patients presenting with a clinical course of either MD or SFD. Nevertheless, a much higher frequency of CD4 T cells committed to cell death was observed in patients with SFD, suggesting a relationship between functional anergy and apoptotic commitment of CD4 T cells during severe H1N1v infections.

Lymphocyte apoptosis and functional anergy could be the result of virus-induced cytokine stimulation, viral induction of Fas/FasL, overstimulation of the immune system, or other mechanisms. We analyzed the plasma levels of inflammatory cytokines and chemokines with the aim of verifying whether a specific set of soluble factors could be associated with a severe clinical course. Although such inflammatory cytokines as IL-8 and IL-6 were detected during H1N1v infection, no major cytokine storm, as seen in H5N1 infection, was observed, confirming recent data [41]. Nevertheless, the clinical course of SFD was associated with a tremendous (40-fold) reduction in the levels of circulating IFN-α and with a significant (1.62-fold) reduction in levels of MCP-1.

IFN-α plays a dominant role in the innate response to influenza [25–27], and it is known that influenza viruses have evolved active mechanisms to escape the host antiviral cytokine response [21] by blocking host IFN genes. Although from the present data it is not possible to establish the functional state of the main natural IFN-α-producing cells (plasmacytoid dendritic cells) in patients with SFD, it is reasonable to hypothesize that the general immune deficiency observed in such patients could play a role in the defective activation of IFN-α.

MCP-1 is a chemokine responsible for monocyte recruitment in the infected tissues, and this migration plays an essential role in innate immune response to influenza [42]. In a mouse model of influenza infection, the blocking of monocyte/macrophage recruitment in the lung resulted in enhanced alveolar epithelial damage and apoptosis [43]. Plasma MCP-1 levels are significantly reduced in patients with SFD, suggesting that reduced MCP-1 levels may be involved in the mechanism of impaired recruitment of monocytes and macrophages in lung tissue.

We are aware of the intrinsic limitations of possible results inferred from our sample, because of sample size and clinical variability. Nevertheless, our results showed, for what we believe is the first time, an association between the immunological host competence and the clinical severity of H1N1v infection, and further studies are warranted to establish the exact involvement of viral and/or host factors in priming this immunological paralysis. The profound impairment of T-cell response induced at least partially by apoptotic commitment exposes the host to viral-induced damage and to secondary infections [44]. In addition, our observation that IFN-α and MCP-1 levels are profoundly impaired in patients with SFD depicts a general deterioration of both innate and adaptive response in these patients, suggesting an association between immune anergy and rapid insult with significant pulmonary damage.

On the whole, the present findings, despite the still unravelled underlying mechanisms, may form the basis for a preemptive approach to monitor host functional response, to identify in a timely manner patients who have an enhanced risk of developing influenza-associated severe complications.

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Acknowledgments

We thank Giulia Berno for technical support. This study is dedicated to Fabrizio Poccia, a great scientist who died at the age of 39 years.

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Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: Italian Ministry of Health “ Ricerca Corrente-Istituti di Ricovero e Cura a Carattere Scientifico” (institutional grant) and Istituto Superiore di Sanità (grant 28C5).

  • Received January 12, 2010.
  • Accepted March 31, 2010.
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  1. J Infect Dis. (2010) 202 (5): 681-689. doi: 10.1086/655469
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