ANÁLISIS COMPARATIVO, EVOLUCIÓN, TENDENCIA Y

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ABSTRACT

Introduction: Aspergillosis occurs in 20-25% of influenza patients at the ICU, suggesting that

an immune defect may account for this increased susceptibility to Aspergillus fumigatus. In bacterial sepsis, systemic immunosuppression called “immunoparalysis” often develops, which is associated with the development of secondary infections.

Methods: In the current study, we studied various aspects of the immune response in

patients with severe influenza infections that were admitted to the ICU to elucidate whether influenza induces systemic immunoparalyis that could explain the increased susceptibility to Aspergillus fumigatus infections. We compared these patients with patients suffering from bacterial pneumonia, isolated neurotrauma, healthy individuals, and healthy volunteers who were intranasally challenged with the live-attenuated influenza vaccine Fluenz.

Results: Circulating cytokine responses, cytokine production upon ex vivo stimulation of

PBMCs with various ligands and populations of IFNγ-, IL-17A-, and IL-22-producing T-cells were not altered in influenza patients, compared with the control groups. Also, in healthy volunteers challenged with Fluenz, no effects on ex vivo cytokine production were observed.

Conclusions: Severe influenza infection does not result in systemic immunosuppression

that may explain the increased susceptibility to Aspergillus fumigatus. Additional studies are crucial to elucidate the factors underlying influenza-induced aspergillosis.

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INTRODUCTION

Influenza infection is generally a self-limiting disease without major complications. However, it may result in an uncontrolled detrimental hyperinflammatory immune state(1)_{. Intriguingly,}

patients may also exhibit hallmarks of profound immunosuppression(2)_{, associated with}

an increased incidence of secondary infections. Bacterial co-infections caused by, e.g.,

Staphylococcus aureus or Streptococcus pneumoniae are frequently observed in influenza

patients(3)_{, but also infections caused by the opportunistic pathogenic fungus Aspergillus} fumigatus have recently been recognized as a significant complication of influenza infection(4, 5)_{. Approximately 20-25% of influenza patients who require admission to the intensive care}

develop invasive pulmonary aspergillosis (IPA), which is associated with high mortality rates(4,

6-11)_{. This high incidence of influenza-associated aspergillosis may suggest that a severe}

influenza infection causes a systemic immune defect that may account for the increased susceptibility toward this particular fungal infection. The characterization of inflammatory responses against Aspergillus fumigatus and other immunogenic stimuli by circulating immune cells of patients may demonstrate such an underlying immune defect.

Following bacterial sepsis, systemic immunosuppression called “immunoparalysis” may develop, which is associated with the development of secondary infections. Immunoparalyis is characterized by functional defects in circulating leukocytes, causing alterations in antigen- presenting ability and cytokine production capacity(12, 13)_{, but also in tissue-resident cells}

obtained from postmortem organ samples of sepsis patients, which show depletion of splenic CD4+, CD8+, and HLA-DR+ _{cells, and increased expression of PD-L1 on various immune cells}(14, 15)_.

In influenza patients, it is not clarified what mechanisms may contribute to the increased susceptibility to secondary infections. Several murine models demonstrate an influenza- induced hyperinflammatory state upon subsequent LPS administration(16)_{. Murine influenza}

infection also results in augmented systemic and pulmonary inflammation, which is associated with enhanced pulmonary neutrophil influx, upon a secondary bacterial infection(17, 18)_.

However, other studies in influenza-infected mice show local immunosuppression upon bacterial co-infection, demonstrated by attenuated pulmonary pro-inflammatory cytokine levels(19, 20)_{, less recruitment and/or impaired functionality of neutrophils, natural killer (NK)-}

cells and tissue-resident macrophages(18, 20, 21)_{, leading to impaired phagocytosis, attenuated}

microbial killing, and impaired antigen presentation. However, whether influenza infection causes systemic immunoparalyis, as observed during bacterial sepsis, remains unknown.

We studied various aspects of the immune response in patients with severe influenza infections who were admitted to the ICU, to elucidate whether or not influenza induces systemic immunoparalyis that could explain the increased susceptibility to Aspergillus fumigatus infections. We compared these patients with other patients admitted to the ICU without influenza, such as patients suffering from bacterial pneumonia or isolated neurotrauma, healthy individuals, and volunteers who were intranasally challenged with Fluenz, a live- attenuated influenza vaccine (LAIV), used as a model for an actual influenza infection.

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Study design

The study was carried out in the Netherlands in accordance with the applicable rules concerning the review of research ethics committees and informed consent. All legal representatives were informed about the study details. All procedures complied with the Declaration of Helsinki including current revisions and the Good Clinical Practice guidelines. A total of 7 adult influenza patients who were admitted to the intensive care department of the Radboud University Medical Center in Nijmegen during the influenza seasons of 2012-2016, were included. In this period, only patients with influenza A (H1N1>H3N2), by far the most common influenza type, were included. Influenza infection was diagnosed by strong clinical suspicion and/or positive Polymerase Chain Reaction (PCR) measured in bronchoalveolar lavage (BAL) fluid. Diagnosis of influenza-associated aspergillosis was made by positive influenza PCR and a galactomannan measurement in the blood of ratio >0.5 and/or BAL of ratio >1.0, as described previously(22)_{. Candida spp. were assessed by positive antigen in blood}

and Pneumocystis spp. were assessed by positive specific PCR in BAL fluid. Viral reactivation of cytomegalovirus (CMV) was determined by PCR in BAL fluid as described previously(23). Bacterial pneumonia was defined as a strong clinical suspicion of bacterial pneumonia and/or positive bronchial / sputum culture for Staphylococcus aureus / Streptococcus

pneumoniae, or positive PCR for other bacterial pathogens in BAL fluid. In total, 7 patients

with bacterial pneumonia, 6 patients with an isolated neurotrauma, and 10 healthy controls were included as controls. The local ethics committee of the Radboud university medical center approved the blood donation for the healthy volunteers (CMO 2010/10). Arterial blood from all patients was drawn from an arterial catheter that was already in place within 24 hours of ICU admission and venous blood was obtained from the healthy volunteers who provided written informed consent. Blood was used for assessment of circulating cytokines and ex

vivo cytokine responses of peripheral blood mononuclear cells (PBMCs). Also, populations of

IFN-γ-, IL-17A-, and IL-22-producing T-cells were measured by intracellular staining followed by flow cytometry. In addition, to further explore the effects of influenza infection on innate immunity, we measured ex vivo cytokine responses of PBMCs obtained from 15 healthy volunteers who were intranasally challenged with Fluenz, a live-attenuated influenza vaccine (LAIV) used as a model for an actual influenza infection in the context of an earlier study from our group (NCT02642237(24)). In this study, blood was sampled at baseline, 2, 14 and 29 days after Fluenz inoculation. Details of the Fluenz challenge study are provided elsewhere(24).

Ex vivo PBMC stimulation

PBMCs were isolated from ethylenediaminetetraacetic acid (EDTA)-anticoagulated blood as described previously(25)_{. Cells were counted using a particle counter (Beckmann}

Coulter, Woerden, The Netherlands) and cell numbers were adjusted to 5×106_{/mL. PBMCs}

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