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# Evaluating the evidence for GM-CSF as a host-directed therapy in respiratory infections

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

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Camille David, Charles Verney, Mustapha Si-Tahar, Antoine Guillon. Evaluating the evidence for GM-CSF as a host-directed therapy in respiratory infections. Cytokine, 2025, 189, pp.156902. 10.1016/j.cyto.2025.156902. hal-04993606

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# Evaluating the Evidence for GM-CSF as a Host-Directed Therapy in Respiratory Infections

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" />

<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**

Novel therapeutic approaches are needed to treat respiratory infections due to the rising antimicrobial resistance and the lack of effective antiviral therapies. A promising avenue to overcome treatment failure is to develop strategies that target the host immune response rather than the pathogen itself. Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a critical role in controlling homeostasis in lungs, alveolar macrophages being the most sensitive cells to GM-CSF signaling. In this review, we discuss the importance of GM-CSF secretion for lung homeostasis and its alteration during respiratory infections. We also present the pre-clinical evidence and clinical investigations evaluating GM-CSF-based treatments (administration or inhibition) as a therapeutic strategy for treating respiratory infections, highlighting both supporting and contradictory findings.

**Keywords**

Granulocyte-macrophage colony-stimulating factor, respiratory infection, influenza virus, SARS-CoV-2 virus, community acquired pneumonia.
# 1. Introduction

Acute respiratory infections are responsible for more than 4 million deaths annually and rank as the third leading cause of death globally, following heart disease and stroke [1,2]. Pneumonia is an infection of the respiratory portion of the lungs, leading to the densification of alveolar structures filled with inflammatory exudates and degraded cell products. It could be divided into two entities: (i) pneumonia acquired outside healthcare units, named community-acquired pneumonia (CAP), and (ii) healthcare-associated pneumonia. These two types of pneumonia differ regarding causal pathogens and patient types; CAP is the main focus of this review. CAP is a public health issue, associated with significant morbidity, mortality and cost. It accounts for 3-5 cases for 1,000 person-years and is the leading infectious reason for admission in emergency care units [3]. The most common pathogens identified in adults with CAP are human influenza virus, rhinovirus, SARS-CoV-2 (recently) and *Streptococcus pneumoniae* [4]. Tuberculosis, a subclass of CAP caused by *M. tuberculosis*, is among the most common infectious diseases and a frequent cause of death worldwide, especially in sub-Saharan countries. However, it is not discussed here because the physiopathology is drastically different than classical CAP. Pneumonia is a primary cause of sepsis and acute respiratory distress syndrome (ARDS). Sepsis is defined as an aberrant or dysregulated host response to infection and the presence of organ dysfunction [5]. ARDS is a clinical syndrome of life threatening acute hypoxemic respiratory failure due to lung inflammation, not caused by cardiogenic pulmonary edema [6].

While antibiotics have significantly improved the treatment of respiratory infections, their effectiveness is waning due to the rise of antibiotic-resistant infections. Today, antimicrobial resistance is considered one of the most serious health threats [7], with nearly 5 million deaths worldwide in 2019 [8]. Regarding virus-related CAP, the need for treatment is obvious. There is a strong skepticism regarding the real efficacy of approved anti-influenza drugs [9,10], and there are no specific treatments for acute infections caused by rhinoviruses [11]. Additionally, there is no strong recommendation for anti-viral therapies to treat severe COVID-19 cases [12]. A promising avenue to overcome treatment failure is to develop novel therapeutic approaches on the basis of specific factors identified in the host. Recent progress in understanding host-pathogen interactions indicates that within minutes to hours of
infection, innate immunity can be induced in any tissue. This activates specific immune cells and the production of an array of antimicrobial signaling pathways and effectors for rapid defense against a broad spectrum of pathogenic microorganisms. Host-directed therapy refers to therapies that target the host immune response rather than the pathogen itself. It has the potential, for pneumonia, to improve outcomes, enhance the efficacy of traditional treatments, and paves the way for new therapies. Host-directed therapy targeting alveolar macrophages could be an efficient strategy during respiratory infections. Alveolar macrophages (AM) are crucial in the defense against respiratory infections as they are the first line of innate immune defense in the alveoli of the lungs (see review [13,14]). Patrolling alveolus, they engulf and destroy pathogens through phagocytosis [15], produce inflammatory mediators to recruit other immune cells, and present antigens to T cells to initiate adaptive immunity [16]. Their ability to rapidly respond to and neutralize pathogens helps preventing the spread of infection and maintain pulmonary health. Additionally, they play a role in clearing debris and apoptotic cells, ensuring the alveolar space remains clean for efficient gas exchange. By boosting the function of alveolar macrophages, such host-directed therapies can improve the clearance of pathogens, reduce inflammation, and limit tissue damage.

GM-CSF is a colony-stimulating factor that acts as a cytokine and is essential for the survival and proliferation of AM, ensuring a sufficient population to effectively defend against respiratory pathogens [17,18]. It also plays a pivotal role in the maturation and activation of AM, enhancing their phagocytic capacity and ability to produce inflammatory mediators. Consequently, GM-CSF has recently been considered as therapy to treat respiratory infection.

Below, we discuss the importance of GM-CSF for AM homeostasis functions, the current knowledge on altered GM-CSF production during respiratory infection and the pre-clinical and clinical evidence that support GM-CSF administration as potential host-directed therapy in the context of pneumonia.

## 2. Importance of GM-CSF Secretion for Lung Homeostasis Controlled by AM

Originally defined as a major myeloid growth factor, GM-CSF (CSF2 gene) was subsequently found to have important effects on mature myeloid cells, such as macrophages and neutrophils [19]. GM-CSF is produced locally in tissues by a
variety of cells including epithelial cells, monocytes/macrophages, T-cells and fibroblasts, and is virtually undetectable in the systemic circulation in the absence of inflammation [20]. GM-CSF binds and activates the GM-CSF receptor, which is a type I cytokine receptor. The GM-CSF receptor is composed of two subunits: an alpha subunit (GM-CSFRα, also known as CD116, CSF2RA gene) and a beta subunit (GM-CSFRβ, also known as CD13, CSF2RB gene). Upon binding of GM-CSF to its receptor, signalling is initiated through the activation of various intracellular pathways, such as *Janus kinase/signal transducer and activator of transcription (JAK-STAT)*, *mitogen-activated protein kinases (MAPK)* and *phosphoinostide 3-kinase (PI3K)* [21]. The pleiotropic effects of GM-CSF on monocytes, macrophages and neutrophils range from increased cell proliferation and survival to enhanced proliferation, phagocytosis, bacterial killing and increased macrophage cytotoxicity [22].

AM originate from fetal liver monocytes that migrate to the lungs during embryonic development, maturing fully around the time of birth. GM-CSF is essential for AM differentiation; however, and unlike most tissue macrophages, AM also depend on transforming growth factor-β receptor (TGF-βR) signaling. In adult mice, both GM-CSF and TGF-β remain crucial for maintaining AM homeostasis [17,23–27]. GM-CSF is mostly produced by type II alveolar epithelial cells (AECs) within the alveolar microenvironment. AM themselves are the source of TGF-β, suggesting an autocrine loop that supports their self-maintenance [23]. GM-CSF signaling involves the regulation of *spi-1 proto-oncogene* (PU.1) and *peroxisome proliferator-activated receptor gamma* (PPARγ) that are key transcription factors necessary for AM development [18,28,29].

Cross-talk between AM and AECs is essential for maintaining lung homeostasis [30]. At steady state, AM exhibits a high basal expression of CD200R, *programmed cell death 1* (PD-1) and *signal-regulatory protein alpha* (SIRPα), which interact with their corresponding AEC ligands CD200, PDL-1, and CD47, respectively to balance inflammatory responses and quiescence [30–35]. AM and AEC communication also contributes to alveolar repair processes. *Tumour necrosis factor alpha* (TNFα) released by activated AM induces epithelial GM-CSF expression, which in turn initiates AEC proliferation and contributes to restoring alveolar barrier function [36].

AM are immersed in pulmonary surfactant, a complex mixture of lipids and proteins that coats the inner surface of the alveoli and reduces surface tension to prevent
alveolar collapse. GM-CSF plays a crucial role in maintaining the proper function of AM, indirectly influencing the catabolism and recycling of surfactant. The breakdown products processed by AM are recycled by type II AECs to generate new surfactant. The presence and function of AM, influenced by GM-CSF, ensure that surfactant is continuously renewed and balanced, which is vital for efficient gas exchange. A rare lung disease caused by GM-CSF deficiency and named pulmonary alveolar proteinosis (PAP), highlights the importance of the GM-CSF regulation in surfactant metabolism or clearance [37–39]. The GM-CSF deficiency in PAP is primarily caused by the presence of autoantibodies against GM-CSF, accounting for 90% of all cases, or by mutations in CSF2RA and CSF2RB genes for other etiologies [40–42]. A *csf2rb*⁻/⁻ mice model reproduce the features of PAP [43,44]. In addition, secondary PAP (by opposition of autoimmune or congenital PAP) can occurs in certain infections (e.g. SARS-CoV-2 or *Nocardia* infection) or chronic exposure to inhaled substances (e.g. silica) [45–48]. All etiologies lead to reduced lung surfactant clearance, accumulation of surfactant components in the airways, and ultimately, impaired gas exchange with respiratory failure [38,40].

### 3. Altered GM-CSF Secretion during Acute Respiratory Infections

While the role of GM-CSF in maintaining homeostasis within the alveolar microenvironment is quite clear, analyzing its altered production during lung infections is complex. AM have a dual role: they are essential for the initial defense against lung infections through pathogen recognition, phagocytosis, and cytokine/chemokines production (e.g. TNF-α, IL-1, IL-6, CCL2, CXCL8); additionally, they regulate inflammation to prevent excessive tissue damage. They secrete anti-inflammatory cytokines like IL-10 and TGF-β and they limit the leukocyte infiltration into the alveolus by efferocytosis of activated neutrophils and monocytes [49–51]. Consequently, the level of GM-CSF secretion during lung infection could be interpreted as either sub-optimal for promoting AM function and survival, or on the contrary, as deleterious if it leads to an AM mediated-uncontrolled inflammation (e.g. cytokine storm) [52]. These interpretations may also be dependent of the inflammatory models used [36].

There is little doubt that GM-CSF signaling impairments before the onset of infections decreases the lung's defense capacities. GM-CSF-deficiency in mice induce more
susceptibility to pulmonary *S. pneumoniae* or Influenza virus infection that can be
corrected by GM-CSF treatment [53–56]. Patients with acquired deficiency in GM-
CSF are susceptible to infections with *Cryptococcus neoformans* and other
opportunistic fungi [57–59]. Similarly, GM-CSF deficient mice infected with
*Cryptococcus neoformans* have a decrease recruitment and activation of
macrophages at the alveolar site resulting in increased susceptibility to the infection
[59].

Lung and circulating levels of GM-CSF are rarely measured during lung infections.
To our knowledge, GM-CSF levels in humans with influenza infection remain
unreported. In the context of SARS-CoV-2 lung infection, either absence or moderate
increased levels of circulating GM-CSF were observed in patients with COVID-19
[45,60,61].

Of note, the blood concentration of GM-CSF in healthy individuals is limited ranging
from 2 to 15 pg/mL. In mice, serum concentration of GM-CSF was significantly
decreased 7 days after influenza A infection (from 30 to 20 pg/mL) [62]. Interestingly,
Bosteels *et al*. identified a GM-CSF-dependent macrophage signature in the lungs of
COVID-19 patients. They also observed that mononuclear cells in these patients
lacked the evolutionarily conserved GM-CSF instruction, resulting in
monocyte/macrophage populations typically seen in GM-CSF-deficient conditions
[45].

Overall, the absence or only moderate increase in GM-CSF levels in COVID-19
patients appears inadequate to support the heightened demand for AM needed to
restore disrupted alveolar homeostasis during SARS-CoV-2 lung infection. However,
since disease severity in COVID-19 is associated with the dysregulated release of
multiple cytokines in later stages, some authors have drawn parallels between
COVID-19 and Cytokine Release Syndrome (CRS) triggered by chimeric antigen
receptor (CAR) T-cell therapy. As a result, inhibiting GM-CSF signaling has been
proposed as a potential strategy to limit excessive infiltration of pro-inflammatory
monocytes in the lungs, thereby preventing cytokine storms and the progression to
ARDS in COVID-19 patients [61].

Nevertheless, the dynamics of GM-CSF expression during acute respiratory
infections and its effect on AM immune responses remain unclear.
4. Pre-clinical Evidence to Support GM-CSF as Therapeutic Strategy during
Respiratory Infections

Influenza A virus (IAV) is a major human pathogen that produces significant morbidity and mortality. Yet, the precise contribution of AM in regulating the severity of IAV infection remains unclear [63]. In 2014, Schneider et al. demonstrated that the absence of AM in GM-CSF-deficient (Csf2-/-) mice or selective AM depletion in wild-type mice resulted in impaired gas exchange and fatal hypoxia associated with severe morbidity to influenza virus infection, while viral clearance was affected moderately [64]. Next, Cardani *et al*. demonstrated that AM deficiency resulted in a marked increase in susceptibility of AECs to IAV infection, resulting in the development of diffuse alveolar damage (the histologic correlate of most patients ARDS) and lethal injury following infection [65]. These observations, along with others [66–69], underscore the critical role of AM in modulating disease severity during influenza virus infection.

To further explore the role of GM-CSF during influenza infection, researchers have
developed mice engineered to continuously secrete high levels of GM-CSF as well
as mice with inducible GM-CSF expression driven by pulmonary-specific promoters
[70,71]. While mice that continuously secrete high levels of GM-CSF developed
desquamative interstitial pneumonia that impaired long-term recovery from influenza,
conditional expression of optimal GM-CSF levels at the time of infection resulted in
enhanced AM proliferation and increase animal survival rate. However, the beneficial
effect was observed only if the GM-CSF expression was triggered before the
infection. Importantly, protection provided by GM-CSF depended on AM, but not on T
cells or B cells [71]. Next, Halstead et al. demonstrated that airway GM-CSF over-
expression starting at 3 days post-influenza infection in GM-CSF-inducible mice,
provided protection against mortality and preserved multiple lung mechanical
properties [72]. Overall, supraphysiological levels of GM-CSF expression in the lung
conferred resistance to influenza infection [55,70,72,73], through an AM-dependent
mechanism [71,72]. The next question from a translational perspective was whether
administering recombinant GM-CSF to the airways after infection onset could offer
protection. In murine models of IAV pneumonia, intratracheal GM-CSF administration
before influenza infection was shown to reduce mortality [74,75]. However, to our
knowledge, therapeutic administration of GM-CSF after infection onset—mirroring
clinical practice—and its impact on influenza replication have not been tested. GM-CSF treatment has been poorly studied for other viral CAP. In respiratory syncytial virus infection in mice, intratracheal administration of GM-CSF prevented the increased susceptibility to respiratory syncytial virus -induced hyperreactivity and inflammation [86][76].

A key contributor to mortality in influenza infection is secondary bacterial infections [77]. Studies in mice suggest that IAV depletes AM, creating a niche for secondary bacterial infections by disrupting early cellular innate immunity in the lungs, ultimately leading to lethal bacterial pneumonia [63,78–81]. Therapeutic administration of GM-CSF after the onset of the influenza infection has been tested to prevent bacterial co-infection. GM-CSF administration partially restored the impaired early bacterial clearance with efficient protection against *S. pneumoniae* [54,78] and *Staphylococcus aureus* [73]. The main attributable effects of GM-CSF in this timeframe were the restoring of the AM lung number and phagocytic functions [78,82–84]. To translate to human practice, a mouse model of inhaled GM-CSF was established. Briefly, mice were infected with IAV and then received aerosolized recombinant mouse GM-CSF twice daily from day 5 to day 9 post-infection. A bacterial co-infection was induced on day 7 post-influenza infection with *S. pneumoniae*, the primary bacterium responsible for bacterial co-infections in human influenza. The authors observed that inhaled GM-CSF significantly improved survival in mice compared to controls [55]. Similar findings supported a protecting role of inhaled GM-CSF in SARS-CoV-2 with *S. pneumoniae* co-infection [82].

GM-CSF is also known to increase the survival of neutrophil and monocyte (phagocytic cells), as well as phagocytosis and bacterial killing [21,85]. Therefore, a broad spectrum of effects can be anticipated for antibacterial defense. Twice daily dose of GM-CSF in mice reduces epithelial cell death and acute lung injury in response to infection with *Klebsiella pneumoniae* [86]. Therapeutic treatment of mice with GM-CSF improved lung protective immunity and accelerated bacterial clearance after pneumococcal challenge [54]. From the best of our knowledge, GM-CSF treatment has been poorly studied for other animal models of bacterial CAP.

Outside of pneumonia, GM-CSF treatment has been poorly studied in animal models of sepsis. As a reminder, sepsis springs from two key factors: first an infection (with pneumonia being the most common cause) and an excessive and harmful host response resulting in organ(s) dysfunction [5]. The cecal ligation and puncture (CLP)
in murine model is the most widely used model for experimental sepsis and is
currently considered as a gold standard in research since it mimics the nature and
evolution of sepsis in humans [87]. Notably, exogenous GM-CSF administration in
this model enhanced the animal survival rate [88–90]. The attributed mechanisms
include improved mucosal integrity reducing microbial translocation, a higher
bacterial killing, increased T cell proliferation, and limited sepsis-induced
immunosuppression.

Overall, recombinant GM-CSF has recently emerged as an interesting host-directed
strategy for promoting a beneficial AM phenotype. Mice have been the main pre-
clinical model for evaluating GM-CSF therapeutic strategy during experimental
respiratory infections. While mouse models are valuable experimental models for
biological questions and/or proof-of-concept studies, certain limitations should be
kept in mind when extrapolating these findings to humans [91]. A comprehensive list
of published pre-clinical investigations involving GM-CSF interventions is presented
in Table 1.
<table><caption>**Table 1. Pre-clinical Investigations with GM-CSF Administration.** Only curative treatments are presented (delivery after the infection).</caption><thead><tr><th></th><th>Study design</th><th>Animal model</th><th>Main Results</th><th>References</th></tr></thead><tbody><tr><td colspan="5"><strong>Influenza infection</strong></td></tr><tr><td>H1N1 Puerto Rico/8/34 and secondary Sp infection, 3 days post-influenza infection</td><td>Intranasal administration of rmGM-CSF, 1 day before and after infection</td><td>BALB/c</td><td>Increase number and function of AM post-infection<br>Trend to decreased bacterial load in bronchoalveolar lavage fluid (BALF)<br>Reduction of 50% of mice developing pneumococcal pneumonia</td><td>[78]</td></tr><tr><td>H1N1 Puerto Rico/8/34 and secondary Sp infection, 7 days post-influenza infection</td><td>Non-invasive aerosolization of rmGM-CSF, twice a day from 5 to 9 days post-infection</td><td>C57BL/6</td><td>Increase survival rate<br>Decrease the presence of bacteria in the bloodstream</td><td>[55]</td></tr><tr><td>H1N1 WSN/33</td><td>Intratracheal administration of rmGM-CSF, 1 day before and the day of infection</td><td>C57BL/6</td><td>Increase survival rate of young and old mice</td><td>[74]</td></tr><tr><td colspan="5"><strong>SARS-CoV-2 infection</strong></td></tr><tr><td>SARS-CoV-2 (2019-nCoV/USA-WA1/2020) and secondary Sp infection, 6 days post-viral infection</td><td>Intranasal administration of rmGM-CSF, 1 day before infection, then at days 1,3,5 and 7 post-infection</td><td>K18-hACE2 mice</td><td>Increase number and function of AM post-infection<br>Decrease of pro-inflammatory cytokines in bronchoalveolar lavage<br>Decrease bacterial lung load</td><td>[82]</td></tr><tr><td>SARS-CoV-2 (WA1/2020WY96)</td><td>Daily intraperitoneal injection of rmGM-CSF for 14 days</td><td>K18-hACE2</td><td>Increase anti-viral antibody<br>Decrease lung viral titer<br>Increase survival rate</td><td>[92]</td></tr><tr><td colspan="5"><strong>Bacterial pneumonia</strong></td></tr><tr><td>Klebsiella pneumoniae strain</td><td>Intratracheal administration of rmGM the day of infection</td><td>TLR4<sup>ps-d</sup><br>BALB/c</td><td>Decrease lung cell apoptosis<br>Decrease systemic bacteraemia</td><td>[86]</td></tr><tr><td>Sp (group 19 strain EF3030)</td><td>Intratracheal administration of rmGM-CSF, 1, 5, 3, 6 or 12h post-infection</td><td>C57BL/6N</td><td>Decrease bacterial load in bronchoalveolar lavage<br>Increase anti-bacterial activity in AM<br>Decrease Sp-induced apoptosis in AM</td><td>[54]</td></tr></tbody></table>
<table><thead><tr><th colspan="5">Sepsis (out of pneumonia)</th></tr></thead><tbody><tr><td>Obese diabetic CLP sepsis model</td><td>Subcutaneous injection of rm GM-CSF every 12 hours for 7 days starting 6 hours after CLP</td><td>C57BL/6</td><td>Increase survival rate<br>Increase neutrophils and monocytes phagocytosis<br>Increase ROS production</td><td>[56]</td></tr><tr><td>CLP sepsis model and secondary Pseudomonas aeruginosa infection, 5 days post-CLP experience</td><td>Hydrodermic injection of rmGM-CSF for 5 days starting 5 days after CLP</td><td>C57BL/6J</td><td>Increase survival rate<br>Decrease bacterial load in bronchoalveolar lavage<br>Enhance bactericidal function of neutrophils</td><td>[90]</td></tr><tr><td>CLP sepsis experience 10 days after burn injury</td><td>Subcutaneous injection or intraperitoneal administration of highly purified, endotoxin-free mouse GM-CSF on days 5 to 9 after CLP</td><td>A/J mice</td><td>Increase survival rate<br>Restoration of T cell proliferation and interleukine-2 production</td><td>[89]</td></tr></tbody></table>

mouse recombinant GM-CSF: rm GM-CSF; cecal ligation and puncture: CLP; Streptococcus pneumoniae : Sp
## 5. Clinical Evidence Supporting GM-CSF-based Treatments during Respiratory Infections

Despite promising preclinical animal findings, the translation of numerous anti-infectious therapies into clinical practice has been underwhelming, as is often the case in other research areas. Traditional animal models for infection and sepsis typically involve single-sex, single-age cohorts, primarily using mice, which poorly represent the heterogeneous patient populations encountered in clinical settings. Numerous factors contribute to the limited reliability of these models and the frequent failures in translating preclinical findings to critical care. These factors include pathogen specificity to certain hosts, the sterile environment in which laboratory animals are raised, and the absence or minimal use of organ support therapies (e.g. mechanical ventilation or fluid resuscitation) [91]. Consequently, the path from bench to bedside in developing recombinant GM-CSF as a treatment for respiratory infections remains full of obstacles. A detailed list of published and registered clinical trials is provided in Table 2.

### Influenza Virus Infection.

Strategies to combat influenza have predominantly concentrated on developing antiviral medications and boosting the adaptive immune response through vaccination. However, there is limited information on leveraging innate immunity for influenza protection. Preclinical studies in mouse models suggest that GM-CSF may be effective in treating influenza, prompting interest in its potential use in humans [93]. To date, no randomized controlled trials have assessed GM-CSF as an adjunct therapy for influenza pneumonia. While GM-CSF has been explored as an adjuvant for influenza vaccines, this falls outside the scope of this review [94]. Finally, and to paraphrase Waterer Grant: "GM-CSF and influenza : will saving mice help humans?" [95]. This question remains unresolved.

### SARS-CoV-2 Infection

The question of whether pulmonary immune responses to SARS-CoV-2 infection are beneficial or detrimental remains unresolved. Balancing the management of lung inflammation with direct antiviral treatment may be crucial, but the optimal timing and
specific immune targets are not yet clearly defined [96]. Research by Bosteels et al. highlighted that COVID-19 is characterized by impaired GM-CSF-dependent alveolar macrophage instruction and an increase in pro-inflammatory macrophages [45]. They explored the safety and efficacy of inhaled GM-CSF in hospitalized COVID-19 patients with acute hypoxic respiratory failure [45]. Inhalation was preferred over systemic administration due to the localized nature of COVID-19 infection in the lungs [97]. However, continuous comparisons revealed no significant improvement in the PaO₂ gradient or overall oxygenation with inhaled GM-CSF compared to standard care. Additionally, no notable differences were observed in outcomes such as hospital stay duration, progression to mechanical ventilation or ARDS, or all-cause mortality at 4 weeks post-randomization [45]. These findings have been corroborated by subsequent studies [98]. Nonetheless, an improvement in oxygenation was noted in patients with moderate hypoxemia who received inhaled GM-CSF twice daily for 5 days [98]. Currently, six clinical trials are underway to evaluate GM-CSF administration (either inhaled or intravenous) in COVID-19 patients (Table 2).

Conversely, another perspective on GM-CSF manipulation in SARS-CoV-2 infection has emerged. COVID-19 has been likened to cytokine storm syndrome, by analogy with the cytokine release syndrome [99]. This analogy gained traction early in the pandemic, despite some skepticism [96,100,101], leading to the exploration of GM-CSF blockade as a strategy to manage hyperinflammation associated with COVID-19 [102]. A single-center, open-label, non-randomized study assessed the efficacy and safety of mavrilimumab, a monoclonal antibody targeting GM-CSFα to disrupt downstream signaling [103]. Among 13 patients treated with mavrilimumab, there were no deaths within 28 days. Based on these preliminary findings, five clinical trials are now investigating GM-CSF blockers in COVID-19 patients (Table 2).

## Community-Acquired Pneumonia (not influenza or SARS-CoV-2 viruses)

In 1989, Wing at al. demonstrated that GM-CSF administration enhances monocyte cytotoxicity and secretion of TNF-alpha and interferon in patients [85]. GM-CSF administration (sargramostim) was approved by US FDA in 1991 to accelerate bone marrow recovery in allogeneic hematopoietic cell transplant recipients [104]. It was afterward approved in patients receiving bone marrow-suppressive drugs and in persons with post-transplant graft-failure. Considering the potential benefits of
inhaled GM-CSF in enhancing AM activity, there is a rationale for using GM-CSF in the treatment of patients with CAP whatever the pathogen(s) involved. CAP include an important diversity of bacterial and/or viral infections [105,106]. GM-CSF increases monocyte killing of S. aureus and C. albicans *in vitro* in patients with solid cancers receiving chemotherapy [107]. Various case reports have been reported using GM-CSF infusion to stimulate immune function in patients with refractory bacterial and fungal infections [108]. To our knowledge, no clinical investigations reported the use of GM-CSF in CAP, outside of SARS-CoV-2 infection. An ongoing phase I study (NCT02601365) is assessing a single dose of inhaled GM-CSF in respiratory virus-associated pneumonia (respiratory syncytial virus, influenza A/B, parainfluenza 1/2/3, human metapneumovirus, adenovirus, rhinovirus).

## Pneumonia-Related Sepsis

Sepsis is associated with dysregulated innate immunity, which can contribute to its complex pathology [5]. To address this, various immunomodulatory strategies have been developed to counteract the associated immunosuppressive state, with GM-CSF being a notable candidate due to its potential to enhance myeloid cell functions in septic patients.

Despite this potential, GM-CSF therapy has not shown a significant reduction in in-hospital mortality or all-cause mortality at 28 days in adult patients with sepsis compared to placebo [109]. Nevertheless, clinical trials have demonstrated that GM-CSF administration is associated with a decreased rate of infectious complications, improved microbiological resolution, and better blood gas exchange [110–112]. Additionally, GM-CSF has been shown to restore human leukocyte antigen DR (HLA-DR) expression and enhance TNFα production following LPS challenge, indicating its biological efficacy [110, 113, 114]. Decreased HLA-DR expression is widely recognized as a marker for immunosuppression in critically ill patients [115, 116].

Given this, GM-CSF therapy may not achieve significant mortality benefits if administered without considering the individual patient's specific immunological state. Recent studies have explored a precision medicine approach by administering GM-CSF to septic patients with identified immunosuppression, characterized by low HLA-DR levels. While this approach demonstrated the feasibility of HLA-DR-guided
precision medicine, it did not result in a significant improvement in ICU-acquired
infections, which was the primary outcome [117].

Pneumonia-Related ARDS

ARDS is characterized by a severe inflammatory response in the lungs, resulting in increased vascular permeability, alveolar damage, and fluid accumulation in the alveoli. This cascade of events impairs gas exchange and can lead to respiratory failure [118]. In this context, GM-CSF level in BALF fluids have been linked to improved outcome in ARDS patients of various etiologies [119]

GM-CSF has been evaluated in a randomized, double-blind, placebo-controlled trial involving 130 individuals with ARDS. Participants received either intravenous GM-CSF or placebo for 14 days. The study found no significant difference in ventilator-free days between the two groups, which was the primary outcome measure [120]. Additionally, an observational study assessed inhaled GM-CSF as a compassionate treatment in six patients with pneumonia-associated ARDS. While improvements in oxygenation were observed, the study's small sample size and lack of a control group limit the robustness of these findings [121].
<table><caption>Table 2. Clinical Trials with GM-CSF-based Interventions (GM-CSF Administration or Inhibition).</caption><thead><tr><th>Intervention</th><th>Study design</th><th>Patients</th><th>Primary outcome</th><th>References</th></tr></thead><tbody><tr><td colspan="5"><strong>Influenza infection</strong></td></tr><tr><td colspan="5">none</td></tr><tr><td colspan="5"><strong>SARS-CoV-2 infection</strong></td></tr><tr><td>Inhaled GM-CSF twice daily for 5 days</td><td>Prospective, multicenter, placebo-controlled, double-blind, randomized, study</td><td>N=75, COVID-19 pneumonia with moderate hypoxemia</td><td>No difference in the number of days to achieve clinical improvement</td><td>[122]</td></tr><tr><td>Inhaled GM-CSF twice daily for 5 days</td><td>Prospective, multicenter, open-label, randomized, study</td><td>N=81, COVID-19 pneumonia and acute hypoxic respiratory failure</td><td>No Improvement in oxygenation</td><td>[45]</td></tr><tr><td>Inhaled GM-CSF twice daily for 5 days</td><td>Prospective, controlled, open-label, randomized, phase II study</td><td>N=122, COVID-19-associated moderate hypoxemia</td><td>Improvement in oxygenation</td><td>[98]</td></tr><tr><td>Inhaled GM-CSF once daily for 5 days.</td><td>Prospective, multicenter, placebo-controlled, double-blind, randomized, phase IIb study</td><td>Mild or moderate COVID-19</td><td>Results posted</td><td>NCT04707664</td></tr><tr><td>Inhaled GM-CSF twice daily for 5 days</td><td>Prospective, multicenter, placebo-controlled, double-blind, randomized, phase II/III study</td><td>COVID-19 associated moderate hypoxemia</td><td>Results posted</td><td>NCT04642950</td></tr><tr><td>Inhaled GM-CSF once daily for 7 days</td><td>Prospective, multicenter, placebo-controlled, double-blind, randomized, study</td><td>COVID-19 pneumonia and acute hypoxic respiratory failure.</td><td>No results posted</td><td>NCT04569877</td></tr><tr><td>Intravenous infusion of GM-CSF once daily for 5 days</td><td>Prospective, single-center, placebo-controlled, double-blind, randomized, study</td><td>COVID-19 pneumonia and acute hypoxic respiratory failure.</td><td>No results posted</td><td>NCT04400929</td></tr><tr><td>Inhaled GM-CSF twice daily for 5 days</td><td>Prospective, multicenter, open-label, randomized, phase II study</td><td>COVID-19 pneumonia and acute hypoxic respiratory failure.</td><td>Results posted</td><td>NCT04411680</td></tr><tr><td>Inhaled GM-CSF twice daily for 5 days</td><td>Prospective, multicenter, open-label, randomized, phase IV</td><td>COVID-19 pneumonia and acute hypoxic respiratory failure.</td><td>Results posted</td><td>NCT04326920</td></tr></tbody></table>
<table><tbody><tr><td>Single intravenous dose of anti-GM-CSF monoclonal antibody</td><td>Prospective, multicenter randomized, multi-arm, multistage, parallel-group, open-label, adaptive, phase 2, proof-of-concept trial</td><td>N= 146, COVID-19 pneumonia and C-reactive protein (CRP) concentrations of 40 mg/L or greater</td><td>Reduce CRP concentration over time</td><td>[123]</td></tr><tr><td>Single intravenous dose of anti-GM-CSF receptor monoclonal antibody</td><td>Prospective, single-center, open-label, not randomized study</td><td>N=13, severe COVID-19 pneumonia</td><td>No difference in 28-day mortality</td><td>[103]</td></tr><tr><td>Single intravenous dose of anti-GM-CSF receptor monoclonal antibody</td><td>Prospective, multicenter, placebo-controlled, double-blind, randomized, phase II study</td><td>COVID-19 pneumonia</td><td>No results posted</td><td>NCT04397497</td></tr><tr><td>Single intravenous dose of anti-GM-CSF receptor monoclonal antibody</td><td>Prospective, multicenter, placebo-controlled, double-blind, randomized, phase II/III study</td><td>COVID-19 pneumonia</td><td>No results posted</td><td>NCT04447469</td></tr><tr><td>Intravenous infusion of anti-GM-CSF monoclonal antibody for 8 days</td><td>Prospective, multicenter, adaptive, randomized, double-blind, placebo-controlled study</td><td>COVID-19-related ARDS</td><td>Results posted</td><td>NCT04351243</td></tr><tr><td>Intravenous infusion of anti-GM-CSF monoclonal antibody</td><td>Prospective, multicenter, placebo-controlled, double-blind, randomized, phase III study</td><td>severe COVID-19 pneumonia</td><td>No results posted</td><td>NCT04351152</td></tr><tr><td>Single intravenous infusion of anti-GM-CSF monoclonal antibody</td><td>Prospective, multicenter, placebo-controlled, double-blind, randomized, phase II/III study</td><td>Severe COVID-19 pneumonia</td><td>Results posted</td><td>NCT04341116</td></tr><tr><td colspan="5">Community-acquired pneumonia (not influenza or SARS-CoV-2 viruses)</td></tr><tr><td>Single dose of inhaled GM-CSF</td><td>Phase I, open label dose escalation study</td><td>Respiratory virus-associated pneumonia (respiratory syncytial virus, influenza A/B, parainfluenza 1/2/3, human metapneumovirus, adenovirus, rhinovirus)</td><td>No results posted</td><td>NCT02601365</td></tr><tr><td colspan="5">Pneumonia-related sepsis</td></tr><tr><td>Continuous intravenous infusion of GM-CSF (or</td><td>Prospective, placebo-controlled, unblind, randomized, study</td><td>N=40, sepsis (30% with pneumonia) without hemodynamic instability or shock</td><td>Up-regulation of the functional markers of inflammation on</td><td>[110]</td></tr></tbody></table>
<table><tbody><tr><td>placebo) for 72h</td><td></td><td></td><td>circulating neutrophils and monocytes</td><td></td></tr><tr><td>Subcutaneous injections of GM-CSF (or placebo) once daily for 8 days</td><td>Prospective, multicenter, placebo-controlled, double-blind, randomized study</td><td>N=38, sepsis or septic shock and sepsis-associated immunosuppression (58% with pneumonia)</td><td>Increase monocytic HLA-DR expression within 24h</td><td>[114]</td></tr><tr><td>Subcutaneous GM-CSF (or placebo) once daily for 4 days.</td><td>Prospective, multicenter, placebo-controlled, single-blind, randomized, phase IIa study</td><td>N=38, sepsis (unknown origin) with impaired neutrophil phagocytic capacity</td><td>No significant difference in the primary outcome (neutrophil phagocytosis 2 days after the first dose of study drug)</td><td>[124]</td></tr><tr><td>Subcutaneous injections of GM-CSF (or placebo) once daily for 5 days</td><td>Prospective, multicenter, placebo-controlled, double-blind, randomized study</td><td>N=98, sepsis or septic shock presenting with sepsis-induced immunosuppression (34% with pneumonia)</td><td>No significant difference observed between groups regarding ICU-acquired infection</td><td>[117]</td></tr><tr><td>Intravenous injection of GM-CSF once daily for 5 days</td><td>Prospective, placebo-controlled, double-blind, randomized, phase II study</td><td>N=18, sepsis (pneumonia referred as the primary diagnosis, unknown percentage)</td><td>Improved blood gas exchange</td><td>[112]</td></tr><tr><td>Intravenous or subcutaneous injection of GM-CSF once daily for 7 days</td><td>Prospective, multicenter open-label study</td><td>Children with sepsis-induced multiple organ dysfunction syndrome and who demonstrate immunoparalysis</td><td>No results posted</td><td>NCT03769844</td></tr><tr><td colspan="5"><strong>Pneumonia-related ARDS</strong></td></tr><tr><td>Intravenous infusion of GM-CSF (or placebo) for 14 days.</td><td>Prospective, multicenter, placebo-controlled double-blind, randomized, phase II study</td><td>N=130, ARDS (32% with pneumonia)</td><td>No difference in ventilator-free days between groups</td><td>[120]</td></tr><tr><td>Inhalation of GM-CSF</td><td>Observational compassionate study</td><td>N=6, pneumonia-related ARDS</td><td>Oxygenation improvement</td><td>[121]</td></tr><tr><td>Inhalation of GM-CSF once daily for 3 days</td><td>Prospective, multicenter, placebo-controlled, double-blind, randomized, phase II study</td><td>N=45, pneumonia-related ARDS</td><td>No results posted</td><td>NCT02595060</td></tr></tbody></table>
## 6. Conclusion

So far, the bench-to-bedside bridge is broken. Despite a solid understanding of GM-CSF's role in lung homeostasis and promising preclinical findings, clinical trials have yet to confirm the efficacy of GM-CSF interventions. The inflammatory response in pneumonia often acts as a double-edged sword, with both beneficial and detrimental effects. This complexity probably explains the surprising observation that opposed GM-CSF interventions (administration or inhibition) have been investigated in the same group of patients (COVID-19) without a clear signal for or against.

Although reporting negative results can be frustrating, it is crucial as it sheds light on the difficulties faced in translating preclinical findings to clinical practice. Such transparency helps refine and strengthen the pathway from laboratory research to clinical application. It raises the question of whether the gap between preclinical and clinical settings is simply too wide in this case. To bridge this gap effectively, several key areas would need further investigation in humans: (i) understanding the kinetics and levels of GM-CSF production under both normal and pathological conditions, including respiratory infections; (ii) elucidating the compartmentalization of GM-CSF in the lungs; and (iii) identifying conditions leading to altered GM-CSF signaling.

Addressing these knowledge gaps could pave the way for more effective therapeutic strategies and improve the translation of GM-CSF research from animal models to human applications.
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