# The deadly dance of alveolar macrophages and influenza virus

Camille David, Charles Verney, Mustapha Si-Tahar, Antoine Guillon

► To cite this version:

Camille David, Charles Verney, Mustapha Si-Tahar, Antoine Guillon. The deadly dance of alveolar macrophages and influenza virus. European Respiratory Review, 2024, 33 (174), pp.240132. 10.1183/16000617.0132-2024. hal-04993624

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The deadly dance of alveolar macrophages and influenza virus

<table><tr><td>Journal:</td><td>European Respiratory Review</td></tr><tr><td>Manuscript ID</td><td>ERR-0132-2024.R2</td></tr><tr><td>Manuscript Type:</td><td>Mini-review</td></tr><tr><td>Date Submitted by the Author:</td><td>09-Aug-2024</td></tr><tr><td>Complete List of Authors:</td><td>DAVID, Camille; University of Tours; INSERM, Centre d'Etude des Pathologies Respiratoires, U1100<br>Verney, Charles; University of Tours; INSERM, Centre d'Etude des Pathologies Respiratoires, U1100<br>Si-Tahar, Mustapha; INSERM, Centre d'Etude des Pathologies Respiratoires, U1100<br>Guillon, Antoine; INSERM, Centre d'Etude des Pathologies Respiratoires, U1100; CHRU de Tours, Service de Réanimation Médicale,F-37044 Tours, France,</td></tr><tr><td>Keyword:</td><td>Alveolar macrophages, Influenza virus, Human, Mouse</td></tr></table>

SCHOLARONE™
Manuscripts
Dear Dr. Renata L. Riha,

Prof. Antoni Torres,

We are pleased by your positive answer regarding our manuscript entitled "The deadly dance of alveolar macrophages and influenza virus" (ID ERR-0132-2024.R1)

As suggested for minor revision, we added a short subsection addressing bacterial infection in the revised manuscript. We kept this paragraph to 10 lines to adhere to the word limit for the mini-review.

"By targeting critical phagocytic cells such as AM, influenza viruses increase the probability of an opportunistic secondary bacterial infection (also called bacterial co-infection), such as those caused by Streptococcus pneumoniae. These secondary bacterial infections are the major cause of mortality associated with influenza [38]. Experimental depletion of AM replicates the increased bacterial burdens observed during influenza pneumonia [39]. Conversely, administering GM-CSF to mice after the onset of influenza infection has been shown to restore the number and phagocytic function of AM in the lungs, which was associated with more effective protection against Streptococcus pneumoniae and improved survival [34, 40, 41]. To the best of our knowledge, no randomized controlled trial has investigated the use of GM-CSF as add-on therapy in the context of influenza pneumonia."

(GM-CSF: granulocyte-macrophage colony-stimulating factor; AM: alveolar macrophages).

We uploaded a clean version of the manuscript, as well as a marked-up copy (red color).

Once again, thank you for considering our manuscript for publication in the European Respiratory Review and I stay at your disposal for further questions.

Sincerely,

Antoine Guillon (corresponding author), on behalf of the co-authors
# MINI-REVIEW

## The deadly dance of alveolar macrophages and influenza virus

Camille David<fnref n="1" />,<fnref n="2" />, Charles Verney<fnref n="1" />,<fnref n="2" />, Mustapha Si-Tahar<fnref n="1" />,<fnref n="2" />, Antoine Guillon<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

## *Correspondence:

Antoine GUILLON, Centre d'Etude des Pathologies Respiratoires, INSERM U1100, Faculté de Médecine - 10, boulevard Tonnellé, 37032 Tours cedex.

antoine.guillon@univ-tours.fr

<footnote n="1">¹INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France</footnote>

<footnote n="2">²Université de Tours, Tours, France</footnote>

<footnote n="3">³CHRU de Tours, Service de Médecine Intensive Réanimation, Tours, France</footnote>
Abstract

Influenza A virus (IAV) is one of the leading causes of respiratory infections. The lack of efficient anti-influenza therapeutics requires a better understanding of how IAV interacts with host cells. Alveolar macrophages (AM) are tissue-specific macrophages that play a critical role in lung innate immunity and homeostasis. Yet, their role during influenza infection remains unclear. First, our review highlights an active IAV replication within AM, despite an abortive viral cycle. Such infection leads to persistent AM inflammation and diminished phagocytic function, alongside direct mitochondrial damage and indirect metabolic shifts in the alveolar microenvironment. We also discuss the "macrophage disappearance reaction" which is a drastic reduction of the AM population observed after influenza infection in mice but debated in humans, with unclear underlying mechanisms. Furthermore, we explore the dual nature of AM responses to IAV infection, questioning whether they are deleterious or protective for the host. While IAV may exploit immunoevasion strategies and induce AM alteration or depletion, this could potentially reduce excessive inflammation and allow for the replacement of more effective cells. Despite these insights, the pathophysiological role of AM during IAV infection in humans remains understudied, urging further exploration to unravel their precise contributions to disease progression and resolution.

Box summarizing “Questions for future research”.

We have provided an overview of AM role in the human airway during influenza infection,
while also pointing out key questions for future research:

* Do influenza viruses infect human AM and, if so, does the viral replication result in infective viral particles?

* What causes the peculiar disappearance of AM, unique among lung leukocytes, during the initial days of influenza infection in mice?

* Is the immune response of AM beneficial or deleterious to the host during influenza infection?
Introduction

Lung infections cause more loss of disability-adjusted life years worldwide than highly-
recognized threats to the public's health such as cancer, heart attacks or strokes [1]. Influenza
virus is one of the leading causes of respiratory infections. In 1918, over 50 million people
succumbed to severe influenza viral pneumonia. Fast forward a century, despite significant
medical advancements, we still face yearly epidemics that lead to 3-5 million severe illness
cases worldwide, resulting in up to 650,000 deaths [2]. Therefore, it is crucial to develop new
antiviral drugs for better treatment of IAV pneumonia. To achieve this, we need a deeper
understanding of how IAV interacts with host cells, especially in the context of IAV-triggered
lung inflammation, which is believed to be the cause of severe "flu" symptoms according to
several studies [3, 4].

Influenza viruses typically start their infection in the respiratory tract. In uncomplicated IAV
infection, the virus replication is confined to the upper respiratory tract and AM are unlikely to
be actively engaged. However, when the respiratory infection extends deeper into the distal
lungs, AM are the most abundant resident leukocytes in the lower respiratory airways and play
a pivotal role in the innate immune response [5]. IAV-infected cells are phagocytosed by AM
for the recognition of viral RNA by various receptors, which lead to the secretion of type I
interferons (IFNs), pro-inflammatory cytokines, eicosanoids and chemokines. Chemokines
produced at the infection site bring in more immune cells, such as neutrophils, monocytes, and
natural killer cells, to the airways. Working alongside AM, these recruited leukocytes (including
NK cells, monocytes and neutrophils) play a crucial role in triggering apoptosis in the infected
cells and/or in removing virus-infected cells.

AM are specialized tissue-specific macrophages critical for innate immunity and maintaining
lung homeostasis. As frontline defenders of the alveoli and airways, they must strike a delicate
balance: providing rapid surveillance and defense against external pathogens while minimizing
inflammation to preserve the integrity of the thin alveolocapillary membrane and ensure
efficient gas exchange.

Comprising 85% to 95% of all cells sampled via bronchoalveolar lavage (BAL) in both mice
and humans in a steady state [5–8], AM inevitably interact with influenza viruses. However,
their precise involvement in human influenza infection remains largely unclear. Some
unanswered AM-related questions should be underscored and discussed, such as: Do
influenza viruses infect human AM and, if yes, is the viral replication leading to infective viral
particles? Why AM are the only lung leukocytes to “disappear” during the first days of influenza
infection in mice? Is the AM immune response beneficial or deleterious for the host during
influenza infection? Moreover, recent advancements in AM biology over the past decade have
revealed long-term changes in "experienced" AM, previously exposed to infections or insults
[9]. This leads to significant AM heterogeneity and variations in immune baseline between
"experienced" and "naïve" organisms [10]. These discoveries challenge the traditional
experimental models and their translational relevance, underscoring the need to integrate them
into studies of host-pathogen interactions between influenza viruses and AM.

AM are likely to be underrated players of lung innate immunity during influenza infection. In
this review, we aim to summarize insight into the role of human AM during influenza infection
to gain a better understanding of influenza virus pathogenesis.

Search strategy

This manuscript is structured as a narrative review. The published data in the area is
nonhomogeneous and not conducive to meta-analysis. In addition, various high-quality
reviews have been published on either respiratory viral infection, on AM or on innate immune
defense toward influenza infection. Here, we focus on overlapping aspects of AM physiology
and IAV infection between animal models and human. To restrict our findings to the state of
the art, the search period covers 1980-2024. To find relevant English-language publications
in peer-reviewed journals, the following databases were interrogated: PubMed, Google and
Scopus and Medline (Ovid). The search algorithm was based on combinations of the following
medical subject headings (MeSH) or keywords: ("Respiratory virus" OR Influenza) AND
("Innate Immunity' OR "Trained Immunity" OR "Innate immune cells" OR Macrophages OR
"alveolar macrophages") AND (lung OR "respiratory tract") AND (("Models, Animal" OR mice)
OR human). We evaluated the search results to ensure they align with the research question
and considered citation tracking to identify additional relevant literature. Fifty papers have been
referenced in this review to follow the instructions for authors (mini-review).

Do influenza viruses infect AM?

As resident lung leukocytes, AM are likely among the first innate immune cell types to
encounter the virus. However, *in vivo* evidence of influenza-infected AM remains limited.
Intracellular viral nucleoprotein was detectable by day two post-infection in murine AM
collected from BAL [11]. Single-cell mapping of host and viral transcriptome in IAV-infected
mice suggested that ~25% of the "mononuclear phagocyte system" were infected cells [12].
Cross-sections from *ex vivo* infected human lung explants also revealed IAV antigen staining
in AM [13]. Despite these important observations, distinguishing between actual infected AM
and macrophages that have engulfed infected cells remains challenging.

To assess if AM are permissive for influenza virus, *in vitro* models were used. Blood-derived
macrophages were found to be susceptible to influenza infection; however, no efficient
influenza virus propagation was detected independently of the viral strain used for infection
[14]. The lack of productive virus replication has been inconstantly observed in these models
[15]. The various conditions used to culture peripheral monocyte-derived macrophages may account for these observed differences. However, it is not clear if any of these macrophage subsets accurately reflect the biological phenotype of AM [15] as they lack essential characteristics such as expression of sialic acid-binding Ig-like lectin-F (Siglec-F), and the specific phenotype imprinted by the lung environment. In 1982, Rodgers et al. demonstrated that ex vivo infections of murine and human AM were permissive to influenza virus but no release of infectious virus was detected [16, 17]. Subsequent studies focusing on the susceptibility of human AM to IAV revealed varying degrees of uncertainty, with some works suggesting that human AM could be infected by IAV [18–20] and that viral replication is active within the infected AM [19–21], while others reported an abortive virus cycle is, resulting in no significant production of influenza neovirions [18, 20, 22] [23]. These disparities underscore the necessity to elucidate the interaction between AM and IAV in humans.

## Changes induced by influenza virus infection on AM metabolism and functions.

Upon contact with influenza virus, one of the earliest AM changes observed is a robust pro-inflammatory response; positioning AM as tissue sentinels orchestrating immune cell recruitment during the initial inflammation phase. In human and porcine AM infected by IAV in vitro, TNF-α stands out as the most secreted cytokine [22, 24], peaking between 4 and 8 hours post-infection. Additionally, AM secrete type I interferon response, a potent antiviral cytokine signal, upon *in vitro* influenza infection [20]. This triggers an amplification loop for viral sensing, evidenced by increased expression of TLR3 and TLR7 at 24 hours post-infection [22, 25]. Despite their pivotal role in initiating inflammation, AM produce fewer inflammatory mediators compared to blood-derived macrophages [26, 27]. After IAV infection, peroxisome-activated receptor gamma (PPAR-γ, a negative regulator of the inflammatory) is downregulated in AM through type I IFN-dependent signaling. PPAR-γ downregulation is associated with persistent inflammation and impaired pneumonia resolution [28]. Furthermore, AM exhibit diminished phagocytic function for several weeks post-influenza infection [29], a phenomenon known as immunoparalysis. This impairment is characterized by altered phagocytosis and decreased expression of key macrophage receptor genes, such as C-type lectin domain family 7 member A (CLEC7A) and macrophage scavenger receptor 1 (MSR1) [22].

Metabolic reprogramming is critical for AM to control cell functions and inflammatory status, with glycolytic shift and modulation of the Krebs cycle being the most important factors [30]. The alveolar environment, rich in oxygen and poor in glucose, appears critical in determining this cellular fate. During severe infections, the lung environment is significantly altered. Inflammation causes the capillaries surrounding the alveoli to become more permeable. This increased permeability causes fluid and proteins to leak into the alveoli, impairing gas exchange. Consequently, there is a significant regional drop in oxygen and an influx of serum
rich in glucose. *In vitro* studies of AM in hypoxic conditions demonstrated the stabilization of HIF1-α that leads to increased glycolysis in human or murine AM [31, 32]. AM have been reported to exhibit distinct cytokine profiles depending on whether their metabolism is centered around oxidative phosphorylation or glycolysis [32]. During *in vivo* influenza infection, it is thus complicated to decipher whether the viral infection induces a glycolytic shift *per se* or if the environmental changes (oxygen deprivation, glucose enrichment) are the main responsive for the metabolic changes. In AM studied outside the alveolar environment, influenza virus infection resulted in an increased accumulation of abnormal mitochondria within the macrophage [33]. Transcriptomic analysis revealed that there was a decreased enrichment of mitochondrial OXPHOS-related genes in *ex vivo* influenza-infected AM [33]. It is likely that influenza infection may cause direct defective mitochondrial fitness and indirect metabolic shift due to altered alveoli environment in the context of infection. A summary of altered cell functions in influenza-infected AM is presented in Fig. 1.

## The “macrophage disappearance reaction”.

During influenza infection, a drastic reduction in the AM population is observed in mice and referred to as the “macrophage disappearance reaction” [9]. This decrease begins within the first 24 hours of infection, peaks at day 7 post infection, and resumes to normal levels by day 10 post-infection [34, 35]. Although variations in the extent of AM depletion post-influenza infection have been noted between mice strains, similar results are observed across various virus strains, independently of viral doses [34, 35]. For example, more than 90% of AM are lost within the first week in BALB/c mice infected with H1N1/PR8 influenza A virus [34]. Studies examining AM pools in both BAL fluids and post-lavage lungs post-infection confirm that the depletion of AM is total, suggesting that influenza-induced AM depletion is not due to upregulated adhesion molecules but rather reflects a genuine loss of AM [34].

To date, the mechanism responsible for AM disappearance in mice remains unknown. It is still unclear whether IAV directly infects AM, leading to cellular death, or if AM disappear due to the loss of survival signals triggered by infection of respiratory epithelial cells (e.g. granulocyte-macrophage colony-stimulating factor [GM-CSF]). Alternatively, AM may migrate outside the airspace during inflammation. IAV possesses an intrinsic ability to subvert and/or suppress the host immune response. This is facilitated by various virulence factors encoded within the virus [36]. One such factor is the polymerase basic protein 1-frame 2 (PB1-F2) which plays a pivotal role in inducing apoptosis in immune cells. PB1-F2 achieves this by targeting mitochondria, leading to significant alterations in their morphology. Consequently, this disrupts the membrane potential, ultimately triggering the apoptotic process [37]. Notably, this effect has been observed in monocytes while its impact on AM remains uncertain. Interestingly, epithelial
cell lines which support productive influenza virus replication, do not exhibit this apoptotic process [37].

By targeting critical phagocytic cells such as AM, influenza viruses increase the probability of an opportunistic secondary bacterial infection (also called bacterial co-infection), such as those caused by *Streptococcus pneumoniae*. These secondary bacterial infections are the major cause of mortality associated with influenza [38]. Experimental depletion of AM replicates the increased bacterial burdens observed during influenza pneumonia [39]. Conversely, administering GM-CSF to mice after the onset of influenza infection has been shown to restore the number and phagocytic function of AM in the lungs, which was associated with more effective protection against *Streptococcus pneumoniae* and improved survival [34, 40, 41]. To the best of our knowledge, no randomized controlled trial has investigated the use of GM-CSF as add-on therapy in the context of influenza pneumonia.

The prevailing concept that IAV infection leads to depletion of AM is widely documented in the literature [34, 42–44]. However, the translational significance of this observation is questionable due to notable differences between human and murine AM. Briefly, human AM display considerable heterogeneity, undergoing constant remodeling during the lifespan, a complexity poorly recapitulated in laboratory mice. Intriguingly, the influenza-induced depletion of AM was not observed in other animal models such as pigs [45] and ferrets [46]. Thus, the “macrophage disappearance reaction” observed in mice has yet to be confirmed in humans and should be interpreted with caution.

## Are AM responses to influenza virus infection deleterious or protective for the host?

The next question is "who benefits from the crime", in this case, the disappearance of macrophages (Fig. 2). On one hand, AM are important for controlling the influenza virus infections and there are clear advantages for influenza viruses in inducing apoptosis in these cells. The recognition of viral PAMPs and nucleic acids by AM receptors triggers an early and strong IFN signaling. Within 4 hours post-infection, seven of the top ten up-regulated genes in IAV-infected AM belong to the type I IFN family [22], essential for combating viral replication [47]. Consequently, macrophages-depleted animal models such as mice [47], ferret [46], pigs [45] and subsequently infected with influenza virus exhibit high virus replication in the airways, more severe respiratory symptoms, and higher mortality rate, compared to infected control animals. Moreover, the influenza mediated depletion of macrophages could hamper antigen presentation to the adaptive immune response, thus reducing the overall ability of the immune system to perform the viral clearance.

On the other hand, this temporary disappearance of AM could also confer advantages to the host. Firstly, during influenza infection, the immune response can become overly aggressive or unregulated, leading to immunopathogenicity [48]. This can damage lung tissues and
contribute to acute respiratory failure [49]. Given that AM are known to sustain persistent
inflammation, their absence could help limit excessive inflammation. Lastly, AM may serve as
decoys for influenza viruses, restricting virus release and spread by undergoing abortive
infection, in contrast to epithelial cells [50]. Therefore, the AM depletion might result from a
"kamikaze" process aimed at preventing infection of surrounding cells. Lastly, there is an
extraordinary heterogeneity among AM in adult humans, including mixed populations of
resident and newly recruited cells [51]. The disruption of homeostasis during influenza infection
contributes to the remodeling of AM populations by recruiting more efficient cells, such as
monocytes that differentiate locally into macrophages or by driving evolution of resident AM
within the lung niche. This process, known as trained immunity, involves the innate immune
system "remembering" past encounters with pathogens, leading to an enhanced response
upon subsequent exposure [52], a phenomenon observed in AM [10]. Interestingly, recent
findings suggest that influenza-trained AM confer long-term antitumor immunity in the lungs
[53].

Conclusions

The pathophysiological significance of AM in IAV infection remains largely unexplored in humans. This gap in knowledge contrasts sharply with the recognized importance of AM in lung biology and the significant public health impact of influenza infection. It is probable that AM play a substantial protective role during IAV infection, which has likely been underestimated thus far. However, direct analysis of AM functions during IAV infection *in vivo* presents several challenges. These include the difficulty in accessing human lung tissues, which necessitates invasive procedures, as well as the limited quantity of AM in IAV-infected mice. Additionally, the situation in human lungs may differ considerably from that of laboratory mice, owing to factors such as repetitive infections and airborne insults, which can influence AM heterogeneity.
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**Contributions:** all authors contributed to the writing of this mini-review and approved the final version.

**Fundings:** the study was supported by the Agence Nationale de la Recherche (ANR-19-CE15-0032-01). CV was funded by "Poste d'accueil INSERM".

**Disclosure statement & competing interests:** no competing interests to declare.
# MINI-REVIEW

## The deadly dance of alveolar macrophages and influenza virus

Camille David<fnref n="1" />,<fnref n="2" />, Charles Verney<fnref n="1" />,<fnref n="2" />, Mustapha Si-Tahar<fnref n="1" />,<fnref n="2" />, Antoine Guillon<fnref n="1" />,<fnref n="2" />,<fnref n="3" />

## *Correspondence:

Antoine GUILLON, Centre d'Etude des Pathologies Respiratoires, INSERM U1100, Faculté de Médecine - 10, boulevard Tonnellé, 37032 Tours cedex.

antoine.guillon@univ-tours.fr

<footnote n="1">¹INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France</footnote>

<footnote n="2">²Université de Tours, Tours, France</footnote>

<footnote n="3">³CHRU de Tours, Service de Médecine Intensive Réanimation, Tours, France</footnote>
**Abstract**

Influenza A virus (IAV) is one of the leading causes of respiratory infections. The lack of efficient anti-influenza therapeutics requires a better understanding of how IAV interacts with host cells. Alveolar macrophages (AM) are tissue-specific macrophages that play a critical role in lung innate immunity and homeostasis. Yet, their role during influenza infection remains unclear. First, our review highlights an active IAV replication within AM, despite an abortive viral cycle. Such infection leads to persistent AM inflammation and diminished phagocytic function, alongside direct mitochondrial damage and indirect metabolic shifts in the alveolar microenvironment. We also discuss the "macrophage disappearance reaction" which is a drastic reduction of the AM population observed after influenza infection in mice but debated in humans, with unclear underlying mechanisms. Furthermore, we explore the dual nature of AM responses to IAV infection, questioning whether they are deleterious or protective for the host. While IAV may exploit immunoevasion strategies and induce AM alteration or depletion, this could potentially reduce excessive inflammation and allow for the replacement of more effective cells. Despite these insights, the pathophysiological role of AM during IAV infection in humans remains understudied, urging further exploration to unravel their precise contributions to disease progression and resolution.

**Box summarizing "Questions for future research".**

We have provided an overview of AM role in the human airway during influenza infection, while also pointing out key questions for future research:

* Do influenza viruses infect human AM and, if so, does the viral replication result in infective viral particles?

* What causes the peculiar disappearance of AM, unique among lung leukocytes, during the initial days of influenza infection in mice?

* Is the immune response of AM beneficial or deleterious to the host during influenza infection?
## Introduction

Lung infections cause more loss of disability-adjusted life years worldwide than highly-recognized threats to the public's health such as cancer, heart attacks or strokes [1]. Influenza virus is one of the leading causes of respiratory infections. In 1918, over 50 million people succumbed to severe influenza viral pneumonia. Fast forward a century, despite significant medical advancements, we still face yearly epidemics that lead to 3-5 million severe illness cases worldwide, resulting in up to 650,000 deaths [2]. Therefore, it is crucial to develop new antiviral drugs for better treatment of IAV pneumonia. To achieve this, we need a deeper understanding of how IAV interacts with host cells, especially in the context of IAV-triggered lung inflammation, which is believed to be the cause of severe "flu" symptoms according to several studies [3, 4].

Influenza viruses typically start their infection in the respiratory tract. In uncomplicated IAV infection, the virus replication is confined to the upper respiratory tract and AM are unlikely to be actively engaged. However, when the respiratory infection extends deeper into the distal lungs, AM are the most abundant resident leukocytes in the lower respiratory airways and play a pivotal role in the innate immune response [5]. IAV-infected cells are phagocytosed by AM for the recognition of viral RNA by various receptors, which lead to the secretion of type I interferons (IFNs), pro-inflammatory cytokines, eicosanoids and chemokines. Chemokines produced at the infection site bring in more immune cells, such as neutrophils, monocytes, and natural killer cells, to the airways. Working alongside AM, these recruited leukocytes (including NK cells, monocytes and neutrophils) play a crucial role in triggering apoptosis in the infected cells and/or in removing virus-infected cells.

AM are specialized tissue-specific macrophages critical for innate immunity and maintaining lung homeostasis. As frontline defenders of the alveoli and airways, they must strike a delicate balance: providing rapid surveillance and defense against external pathogens while minimizing inflammation to preserve the integrity of the thin alveolocapillary membrane and ensure efficient gas exchange.

Comprising 85% to 95% of all cells sampled via bronchoalveolar lavage (BAL) in both mice and humans in a steady state [5-8], AM inevitably interact with influenza viruses. However, their precise involvement in human influenza infection remains largely unclear. Some unanswered AM-related questions should be underscored and discussed, such as: *Do influenza viruses infect human AM and, if yes, is the viral replication leading to infective viral particles? Why AM are the only lung leukocytes to "disappear" during the first days of influenza infection in mice? Is the AM immune response beneficial or deleterious for the host during influenza infection?* Moreover, recent advancements in AM biology over the past decade have revealed long-term changes in "experienced" AM, previously exposed to infections or insults [9]. This leads to significant AM heterogeneity and variations in immune baseline between
"experienced" and "naïve" organisms [10]. These discoveries challenge the traditional experimental models and their translational relevance, underscoring the need to integrate them into studies of host-pathogen interactions between influenza viruses and AM.

AM are likely to be underrated players of lung innate immunity during influenza infection. In this review, we aim to summarize insight into the role of human AM during influenza infection to gain a better understanding of influenza virus pathogenesis.

## Search strategy

This manuscript is structured as a narrative review. The published data in the area is nonhomogeneous and not conducive to meta-analysis. In addition, various high-quality reviews have been published on either respiratory viral infection, on AM or on innate immune defense toward influenza infection. Here, we focus on overlapping aspects of AM physiology and IAV infection between animal models and human. To restrict our findings to the state of the art, the search period covers 1980-2024. To find relevant English-language publications in peer-reviewed journals, the following databases were interrogated: PubMed, Google and Scopus and Medline (Ovid). The search algorithm was based on combinations of the following medical subject headings (MeSH) or keywords: ("Respiratory virus" OR Influenza) AND ("Innate Immunity' OR "Trained Immunity" OR "Innate immune cells" OR Macrophages OR "alveolar macrophages") AND (lung OR "respiratory tract") AND (("Models, Animal" OR mice) OR human). We evaluated the search results to ensure they align with the research question and considered citation tracking to identify additional relevant literature. Fifty papers have been referenced in this review to follow the instructions for authors (mini-review).

## Do influenza viruses infect AM?

As resident lung leukocytes, AM are likely among the first innate immune cell types to encounter the virus. However, *in vivo* evidence of influenza-infected AM remains limited. Intracellular viral nucleoprotein was detectable by day two post-infection in murine AM collected from BAL [11]. Single-cell mapping of host and viral transcriptome in IAV-infected mice suggested that ~25% of the "mononuclear phagocyte system" were infected cells [12]. Cross-sections from *ex vivo* infected human lung explants also revealed IAV antigen staining in AM [13]. Despite these important observations, distinguishing between actual infected AM and macrophages that have engulfed infected cells remains challenging.

To assess if AM are permissive for influenza virus, *in vitro* models were used. Blood-derived macrophages were found to be susceptible to influenza infection; however, no efficient influenza virus propagation was detected independently of the viral strain used for infection [14]. The lack of productive virus replication has been inconstantly observed in these models
[15]. The various conditions used to culture peripheral monocyte-derived macrophages may account for these observed differences. However, it is not clear if any of these macrophage subsets accurately reflect the biological phenotype of AM [15] as they lack essential characteristics such as expression of sialic acid-binding Ig-like lectin-F (Siglec-F), and the specific phenotype imprinted by the lung environment. In 1982, Rodgers et al. demonstrated that ex vivo infections of murine and human AM were permissive to influenza virus but no release of infectious virus was detected [16, 17]. Subsequent studies focusing on the susceptibility of human AM to IAV revealed varying degrees of uncertainty, with some works suggesting that human AM could be infected by IAV [18–20] and that viral replication is active within the infected AM [19–21], while others reported an abortive virus cycle is, resulting in no significant production of influenza neovirions [18, 20, 22] [23]. These disparities underscore the necessity to elucidate the interaction between AM and IAV in humans.

**Changes induced by influenza virus infection on AM metabolism and functions.**

Upon contact with influenza virus, one of the earliest AM changes observed is a robust pro-inflammatory response; positioning AM as tissue sentinels orchestrating immune cell recruitment during the initial inflammation phase. In human and porcine AM infected by IAV in vitro, TNF-α stands out as the most secreted cytokine [22, 24], peaking between 4 and 8 hours post-infection. Additionally, AM secrete type I interferon response, a potent antiviral cytokine signal, upon *in vitro* influenza infection [20]. This triggers an amplification loop for viral sensing, evidenced by increased expression of TLR3 and TLR7 at 24 hours post-infection [22, 25]. Despite their pivotal role in initiating inflammation, AM produce fewer inflammatory mediators compared to blood-derived macrophages [26, 27]. After IAV infection, peroxisome-activated receptor gamma (PPAR-γ, a negative regulator of the inflammatory) is downregulated in AM through type I IFN-dependent signaling. PPAR-γ downregulation is associated with persistent inflammation and impaired pneumonia resolution [28]. Furthermore, AM exhibit diminished phagocytic function for several weeks post-influenza infection [29], a phenomenon known as immunoparalysis. This impairment is characterized by altered phagocytosis and decreased expression of key macrophage receptor genes, such as C-type lectin domain family 7 member A (CLEC7A) and macrophage scavenger receptor 1 (MSR1) [22].

Metabolic reprogramming is critical for AM to control cell functions and inflammatory status, with glycolytic shift and modulation of the Krebs cycle being the most important factors [30]. The alveolar environment, rich in oxygen and poor in glucose, appears critical in determining this cellular fate. During severe infections, the lung environment is significantly altered. Inflammation causes the capillaries surrounding the alveoli to become more permeable. This increased permeability causes fluid and proteins to leak into the alveoli, impairing gas exchange. Consequently, there is a significant regional drop in oxygen and an influx of serum
rich in glucose. *In vitro* studies of AM in hypoxic conditions demonstrated the stabilization of HIF1-α that leads to increased glycolysis in human or murine AM [31, 32]. AM have been reported to exhibit distinct cytokine profiles depending on whether their metabolism is centered around oxidative phosphorylation or glycolysis [32]. During *in vivo* influenza infection, it is thus complicated to decipher whether the viral infection induces a glycolytic shift *per se* or if the environmental changes (oxygen deprivation, glucose enrichment) are the main responsive for the metabolic changes. In AM studied outside the alveolar environment, influenza virus infection resulted in an increased accumulation of abnormal mitochondria within the macrophage [33]. Transcriptomic analysis revealed that there was a decreased enrichment of mitochondrial OXPHOS-related genes in *ex vivo* influenza-infected AM [33]. It is likely that influenza infection may cause direct defective mitochondrial fitness and indirect metabolic shift due to altered alveoli environment in the context of infection. A summary of altered cell functions in influenza-infected AM is presented in Fig. 1.

## The “macrophage disappearance reaction”.

During influenza infection, a drastic reduction in the AM population is observed in mice and referred to as the “macrophage disappearance reaction” [9]. This decrease begins within the first 24 hours of infection, peaks at day 7 post infection, and resumes to normal levels by day 10 post-infection [34, 35]. Although variations in the extent of AM depletion post-influenza infection have been noted between mice strains, similar results are observed across various virus strains, independently of viral doses [34, 35]. For example, more than 90% of AM are lost within the first week in BALB/c mice infected with H1N1/PR8 influenza A virus [34]. Studies examining AM pools in both BAL fluids and post-lavage lungs post-infection confirm that the depletion of AM is total, suggesting that influenza-induced AM depletion is not due to upregulated adhesion molecules but rather reflects a genuine loss of AM [34].

To date, the mechanism responsible for AM disappearance in mice remains unknown. It is still unclear whether IAV directly infects AM, leading to cellular death, or if AM disappear due to the loss of survival signals triggered by infection of respiratory epithelial cells (e.g. granulocyte-macrophage colony-stimulating factor [GM-CSF]). Alternatively, AM may migrate outside the airspace during inflammation. IAV possesses an intrinsic ability to subvert and/or suppress the host immune response. This is facilitated by various virulence factors encoded within the virus [36]. One such factor is the polymerase basic protein 1-frame 2 (PB1-F2) which plays a pivotal role in inducing apoptosis in immune cells. PB1-F2 achieves this by targeting mitochondria, leading to significant alterations in their morphology. Consequently, this disrupts the membrane potential, ultimately triggering the apoptotic process [37]. Notably, this effect has been observed in monocytes while its impact on AM remains uncertain. Interestingly, epithelial
cell lines which support productive influenza virus replication, do not exhibit this apoptotic process [37].

By targeting critical phagocytic cells such as AM, influenza viruses increase the probability of an opportunistic secondary bacterial infection (also called bacterial co-infection), such as those caused by *Streptococcus pneumoniae*. These secondary bacterial infections are the major cause of mortality associated with influenza [38]. Experimental depletion of AM replicates the increased bacterial burdens observed during influenza pneumonia [39]. Conversely, administering GM-CSF to mice after the onset of influenza infection has been shown to restore the number and phagocytic function of AM in the lungs, which was associated with more effective protection against *Streptococcus pneumoniae* and improved survival [34, 40, 41]. To the best of our knowledge, no randomized controlled trial has investigated the use of GM-CSF as add-on therapy in the context of influenza pneumonia.

The prevailing concept that IAV infection leads to depletion of AM is widely documented in the literature [34, 42–44]. However, the translational significance of this observation is questionable due to notable differences between human and murine AM. Briefly, human AM display considerable heterogeneity, undergoing constant remodeling during the lifespan, a complexity poorly recapitulated in laboratory mice. Intriguingly, the influenza-induced depletion of AM was not observed in other animal models such as pigs [45] and ferrets [46]. Thus, the “macrophage disappearance reaction” observed in mice has yet to be confirmed in humans and should be interpreted with caution.

## Are AM responses to influenza virus infection deleterious or protective for the host?

The next question is "who benefits from the crime", in this case, the disappearance of macrophages (Fig. 2). On one hand, AM are important for controlling the influenza virus infections and there are clear advantages for influenza viruses in inducing apoptosis in these cells. The recognition of viral PAMPs and nucleic acids by AM receptors triggers an early and strong IFN signaling. Within 4 hours post-infection, seven of the top ten up-regulated genes in IAV-infected AM belong to the type I IFN family [22], essential for combating viral replication [47]. Consequently, macrophages-depleted animal models such as mice [47], ferret [46], pigs [45] and subsequently infected with influenza virus exhibit high virus replication in the airways, more severe respiratory symptoms, and higher mortality rate, compared to infected control animals. Moreover, the influenza mediated depletion of macrophages could hamper antigen presentation to the adaptive immune response, thus reducing the overall ability of the immune system to perform the viral clearance.

On the other hand, this temporary disappearance of AM could also confer advantages to the host. Firstly, during influenza infection, the immune response can become overly aggressive or unregulated, leading to immunopathogenicity [48]. This can damage lung tissues and
contribute to acute respiratory failure [49]. Given that AM are known to sustain persistent
inflammation, their absence could help limit excessive inflammation. Lastly, AM may serve as
decoys for influenza viruses, restricting virus release and spread by undergoing abortive
infection, in contrast to epithelial cells [50]. Therefore, the AM depletion might result from a
"kamikaze" process aimed at preventing infection of surrounding cells. Lastly, there is an
extraordinary heterogeneity among AM in adult humans, including mixed populations of
resident and newly recruited cells [51]. The disruption of homeostasis during influenza infection
contributes to the remodeling of AM populations by recruiting more efficient cells, such as
monocytes that differentiate locally into macrophages or by driving evolution of resident AM
within the lung niche. This process, known as trained immunity, involves the innate immune
system "remembering" past encounters with pathogens, leading to an enhanced response
upon subsequent exposure [52], a phenomenon observed in AM [10]. Interestingly, recent
findings suggest that influenza-trained AM confer long-term antitumor immunity in the lungs
[53].

Conclusions

The pathophysiological significance of AM in IAV infection remains largely unexplored in humans. This gap in knowledge contrasts sharply with the recognized importance of AM in lung biology and the significant public health impact of influenza infection. It is probable that AM play a substantial protective role during IAV infection, which has likely been underestimated thus far. However, direct analysis of AM functions during IAV infection *in vivo* presents several challenges. These include the difficulty in accessing human lung tissues, which necessitates invasive procedures, as well as the limited quantity of AM in IAV-infected mice. Additionally, the situation in human lungs may differ considerably from that of laboratory mice, owing to factors such as repetitive infections and airborne insults, which can influence AM heterogeneity.
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**Contributions:** all authors contributed to the writing of this mini-review and approved the final version.

**Fundings:** the study was supported by the Agence Nationale de la Recherche (ANR-19-CE15-0032-01). CV was funded by "Poste d'accueil INSERM".

**Disclosure statement & competing interests:** no competing interests to declare.
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<figure><img src="image_3.png" /><figcaption>**Figure 1.** Altered cell functions in influenza-infected AM. C-type lectin domain family 7 member A (CLEC7A), macrophage scavenger receptor 1 (MSR1), CD36, mannose receptor C type 1 (MRC1).</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 2. Hypothetical trade-offs of the “macrophage disappearance reaction” during influenza infection.</figcaption></figure>