# Neutrophil subsets enhance the efficacy of host-directed therapy in pneumococcal pneumonia

Laura Matarazzo, Charlotte Costa, Rémi Porte, Jean-Michel Saliou, Martin Figeac, Fabien Delahaye, Amélie Bonnefond, Benoit Kloeckner, Aymeric Silvin, Florent Ginhoux, et al.

► To cite this version:

Laura Matarazzo, Charlotte Costa, Rémi Porte, Jean-Michel Saliou, Martin Figeac, et al. Neutrophil subsets enhance the efficacy of host-directed therapy in pneumococcal pneumonia. Mucosal Immunology, 2025, 18 (1), pp.257-268. 10.1016/j.mucimm.2024.11.009. inserm-05340629

HAL Id: inserm-05340629

https://inserm.hal.science/inserm-05340629v1

Submitted on 31 Oct 2025

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Distributed under a Creative Commons Attribution 4.0 International License
combining an antibiotic (such as amoxicillin or co-trimoxazole in a sub-
therapeutic dosing) and i.n. administration of flagellin has been found to
be 100-fold more effective in treating pneumonia compared to using the
antibiotic or flagellin alone<sup>16–18</sup>. The combination treatment's superiority
lies in its ability to achieve low bacterial numbers in the lungs, reduce
dissemination, and increase survival rates. Remarkably, this combination

has proven synergistic not only in primary infections but also in the
context of post-influenza superinfections¹⁷. In these models, the admin-
istration of the combined treatment resulted in a local innate immune
signature, characterized by increased gene transcription and production
of chemokines and cytokines, as well as an increased number of neutro-
phils in the airways¹⁷,¹⁸. These findings support the hypothesis that innate

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Respiratory and systemic immune response induced by AMX + flagellin combination therapy during pneumonia. BALB/c mice (n = 4 per group) were infected i.n. with 2 × 10⁶ S. pneumoniae and treated 12 h later by i.g. administration of AMX combined with i.n. administration of flagellin (AMX + flagellin) or PBS (AMX). Lungs, BAL fluids, and serum were sampled at indicated times post-treatment. (A) Gene expression in lungs measured by RT-qPCR. The relative mRNA levels of selected genes are expressed against the reference gene Actb and the reference condition AMX (arbitrarily set to a value of 1). (B-C) Cytokine and chemokine concentration in BAL fluids (B) and serum (C). Statistical differences were analyzed by two-way ANOVA.</figcaption></figure>
immune effectors play a pivotal role in the synergistic protective activity observed with the combined antibiotic + flagellin treatment. However, the precise immune mechanisms driving this synergy remain to be identified. This study aimed to comprehensively analyze the lung to determine the specific immune mechanisms responsible for the therapeutic efficacy of the flagellin-antibiotic combination during intranasal treatment of

pneumococcal pneumonia. To this aim, a dual approach combining functional studies with multi-omic analyses (namely proteomics, transcriptomics and single-cell RNA sequencing) was performed. This integrative strategy allowed to not only validate previous quantitative findings that neutrophil numbers are critical for the protective effect of the combined therapy but also revealed that the efficacy was linked to the

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<table><thead><tr><th rowspan="2">Ingenuity Canonical Pathways</th><th colspan="2">-log(p_value)</th><th rowspan="2">Molecules</th></tr><tr><th>2 h</th><th>4 h</th></tr></thead><tbody><tr><td>Granulocyte Adhesion and Diapedesis</td><td>12.90</td><td>6.82</td><td>C5AR1, CCL11, CCL17, CCL19, CCL20, CCL25, CCL27, CLDN8, CLDN15, CSF3, CXCL2, CXCL3, CXCL6, CXCL12, CXCL13, CXCR4, FPR1, FPR2, IL1B, IL1R2, IL1RN, IL18RAP, IL36A, IL36G, ITGA1, MMP7, MMP8, MMP9, MMP11, MMP25, SDC4, SELE, SELP, TNFRSF1B, TNFRSF11B</td></tr><tr><td>Agranulocyte Adhesion and Diapedesis</td><td>8.50</td><td>4.17</td><td>C5AR1, CCL11, CCL17, CCL19, CCL20, CCL25, CCL27, CLDN8, CLDN15, CXCL2, CXCL3, CXCL6, CXCL12, CXCL13, CXCR4, IL1B, IL1RN, IL36A, IL36G, MMP7, MMP8, MMP9, MMP11, MMP25, MYH7, MYH10, SDC4, SELE, SELP</td></tr><tr><td>Phagosome Formation</td><td>3.9</td><td>6.52</td><td>C5AR1, CLEC7A, CR2, FCER1G, FCGR2A, FCGR2B, FCGR3A/FCGR3B, MRC2, PIK3C2G, PIK3R5, PLCB4, PLCD4, PRKCG, PRKCH, PRKCZ, RAC2, RHOB, RHOC, RHOF, RHQ, RHOU, RHOV, RND1, SYK, Tir13, TLR2, TLR6, TLR7, ARG2, CD14, FCGR2A, FCGR2B, FOS, IKBKE, IL1B, IL1R2, IL1RN, IL10, IL10RA, IL18RAP, IL36A, IL36G, NFKBIA, SOCS3</td></tr><tr><td>IL-10 Signaling</td><td>4.90</td><td>4.07</td><td>APOC2, ARG2, FOS, IKBKE, MAP3K6, MAP3K12, MAP3K8, MPO, NCF1, NCF4, NFKBIA, NOS2, PIK3C2G, PIK3R5, PPP2R2B, PPP1R3C, PRKCH, PRKCZ, RAC2, RHOB, RHOC, RHOF, RHOJ, RHOU, RHOV, RND1, S100A8, TLR2, TNFRSF1B, TNFRSF11B</td></tr><tr><td>Production of Nitric Oxide and Reactive Oxygen Species in Macrophages</td><td>4.50</td><td>4.25</td><td>CD79A, CSF2, CD80, CD83, CSF2, FCER1G, IGHM, IL1B, IL1RN, IL10, IL36A, IL36G, Tlr13, TLR2, TLR6, TLR7, TNFRSF13C</td></tr><tr><td>Communication between Innate and Adaptive Immune Cells</td><td>2.86</td><td>4.75</td><td>CARD11, FCER1G, FGFR4, FLT1, HRAS, IL1B, IL1R2, IL1RN, IL36A, IL36G, IRAK3, MALT1, MAP3K8, NFKBIA, NTRK3, PIK3C2G, PIK3R5, PRKCZ, RASD1, TLR2, TLR6, TLR7, TNFAIP3, TNFRSF1B, TNFRSF11B, TNIP1</td></tr><tr><td>NF-κB Signaling</td><td>4.21</td><td>2.73</td><td>CD14, FOS, IL1B, IL1RN, IL36A, IL36G, IRAK2, IRAK3, NFKBIA, TLR2, TLR6, TLR7, TNFAIP3</td></tr><tr><td>Toll-like Receptor Signaling</td><td>4.57</td><td>1.62</td><td>DUSP1, FAS, HIST1H3C, IL18RAP, IL1B, IL1R2, IL1RN, IL36A, IL36G, IRAK2, IRAK3, MAPT, MYC, TIF1, TNFRSF1B</td></tr><tr><td>p38 MAPK Signaling</td><td>3.77</td><td>2.05</td><td>CD14, FOS, HRAS, IKBKE, IL18RAP, IL1B, IL1R2, IL1RN, IL36A, IL36G, NFKBIA, PIK3C2G, PIK3R5, RASD1, SOCS3, TNFAIP6, TNFRSF1B, TNFRSF11B</td></tr><tr><td>IL-6 Signaling</td><td>4.39</td><td>1.38</td><td>CD80, CD83, COL1A2, CSF2, FCER1G, FCGR2A, FCGR2B, FCGR3A/FCGR3B, IKBKE, IL1B, IL1RN, IL10, IL23A, IL36A, IL36G, NFKBIA, PIK3C2G, PIK3R5, PLCB4, PLCD4, TLR2, TNFRSF1B, TNFRSF11B</td></tr><tr><td>Dendritic Cell Maturation</td><td>3.31</td><td>1.72</td><td></td></tr></tbody></table>

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Transcriptomic analysis of flagellin-induced lung response in pneumococcal pneumonia. BALB/c mice (n = 3–4 per group) were infected i.n. with $2 \times 10^6$ S. pneumoniae and treated 12 h later by i.g. administration of AMX combined with i.n. administration of flagellin (AMX + flagellin) or PBS (AMX). Lungs were collected at 2 h and 4 h post-treatment followed by total RNA extraction. Transcript levels were studied by microarrays (A-B) or RT-qPCR (C). (A) Heat map of the top 30 DEG between AMX + flagellin vs AMX treatments. (B) Enrichment of canonical pathways, according to an Ingenuity Pathway Analysis of DEG with $|log_{2}FC| \ge 1$ and $p \le 0.05$. (C) Validation of signature genes by RT-qPCR in an independent experiment. Statistical differences between AMX + flagellin and AMX groups were analyzed by Mann and Whitney test at each time point. Non-significant differences are indicated by “ns”.</figcaption></figure>
enrichment of distinct neutrophil subsets. This highlights a qualitative effect of the adjunct therapy, suggesting that specific neutrophil subpopulations, rather than total neutrophil counts alone, play a key role in mediating the enhanced protection.

## Results

### *Intranasal administration of flagellin accelerates innate immune response in mice infected with S. Pneumoniae*

To investigate the dynamics of the immune response induced by adjunct flagellin treatment, mice were inoculated i.n. with *S. pneumoniae* and treated 12 h later with a combination of amoxicillin administered i. g. and flagellin FliC<sub>Δ174-400</sub> administered i.n. (AMX + flagellin), or a combination of AMX i.g. and PBS i.n. (referred to as AMX) or a combination of water i.g. and flagellin i.n. (referred to as flagellin) as reference treatments. As described previously<sup>16-18</sup>, the dose of AMX was intentionally selected to be subtherapeutic in order to assess the potential for enhanced efficacy when combined with flagellin. This subtherapeutic dosing allows for an evaluation of the synergistic effects of the AMX + flagellin combination compared to AMX alone or flagellin alone. As expected, analysis of bacterial counts at 12 h post-treatment demonstrated the superiority of AMX + flagellin compared to AMX alone or flagellin alone (Supplementary Fig. 1). All subsequent steps were comparing the following treatment groups: AMX alone and combination AMX + flagellin. We next studied the impact of treatments on cytokine and chemokine expression in the respiratory tract and systemic compartments. Compared to the AMX-treated group, transcription of the immune mediator genes *Ccl20*, *Cxcl2*, *Il6*, and *S100a9* in animals receiving AMX + flagellin peaked at 2 to 4 h post-treatment, and then gradually decreased over time (Fig. 1A). The production of the immune mediators CCL20, CXCL2, IL-6 and TNF was significantly increased in the BAL fluid at 2 h and 4 h post-treatment, and then decreased to low or undetectable levels by 12–24 h (Fig. 1B). Serum levels of CCL20 and CXCL2 exhibited similar kinetics, whereas serum IL-6 was minimally affected by the treatments and TNF was not produced (Fig. 1C). These findings demonstrate that a single administration of the combined treatment AMX + flagellin elicits an immediate and transient innate immune response both in the respiratory tract and the blood.

### *Transcriptional response of lung to the combination therapy reveals phagocyte implication in treatment effectiveness*

To further identify the immune effectors linked to treatment efficacy, an unbiased transcriptomic analysis of the lung was conducted at 2 h and 4 h post-treatment (Fig. 2 and Supplementary File 1). The analysis identified 1205 and 1423 DEG that were significantly regulated (|log<sub>2</sub>FC|≥1 and p ≤ 0.05) at 2 h and 4 h by AMX + flagellin compared to AMX, respectively. Remarkably, upregulated genes were implicated in immune cell activation or recruitment to inflammatory sites (such as chemokines, matrix metalloproteases, formyl-peptide receptors) as well as in antibacterial functions including pentraxin 3, neutrophilic granule protein, acyloxyacid hydrolase, and cathelicidin antimicrobial peptide (Fig. 2A). Pathway analysis revealed a significant modulation of granulocyte and agranulocyte adhesion and diapedesis pathways, along with those related to cell-associated antimicrobial mechanisms, such as phagosome formation and production of nitric oxide and reactive oxygen species (ROS) (Fig. 2B). The expression of selected genes was validated by RT-qPCR in an independent experiment (Fig. 2C). Furthermore, semi-quantitative proteomic analysis of bronchoalveolar lavage (BAL) fluids demonstrated increased levels of proteins at 2 h and 4 h post-treatment that are related to neutrophil and antibacterial functions of neutrophil such as lactotransferrin, lysozyme, lipocalin 2, neutrophilic granule protein, and myeloperoxidase (Supplementary Fig. 2A-B). Higher levels of complement system factors and factors associated with conducting airways physiology were also observed in the AMX +

flagellin group compared to the AMX group. In conclusion, these data underscore the rapid and multifactorial local airway immune response induced by flagellin in the context of ongoing infection, emphasizing the potential role of phagocytes in treatment effectiveness.

### *Flagellin transiently increases phagocyte recruitment in the airways*

We next analyzed the phagocytic cells, i.e., neutrophils, inflammatory monocytes, and alveolar macrophages post-treatment in lungs (Fig. 3). In contrast to AMX, AMX + flagellin administration elicited a significant increase of neutrophil numbers as early as 2 h and 4 h, that was followed by a decrease over time (Fig. 3A). A significant increase of CCR2<sup>+</sup> inflammatory monocytes was also observed in lungs at 4 h in response to flagellin (Fig. 3B). The alveolar macrophage population was not significantly altered at any time or treatment (Fig. 3C). The analysis also showed that the pneumococcal infection by itself stimulated the recruitment of neutrophils and monocytes in the lung compared to naïve uninfected animals but did not affect alveolar macrophages (Fig. 3D-F). The AMX treatment did not modify the number of lung neutrophils and monocytes in contrast to the treatment with AMX + flagellin. Similar findings were observed by analyzing the BAL with regards to neutrophils and alveolar macrophages whereas inflammatory monocytes were almost absent (Supplementary Fig. 3). These data highlight that, even in the context of cell infiltration caused by infection, adjunct flagellin in the setting of antibiotic treatment enhances recruitment of neutrophils and inflammatory monocytes in airways. These experiments raise the hypothesis that an early recruitment of neutrophils and inflammatory monocytes into the lung plays a major role in the synergistic therapeutic effect of adjunct flagellin to antibiotic treatment.

### *Neutrophil recruitment into the airways is crucial for the flagellin-antibiotic therapeutic effect*

To investigate the contribution of neutrophils, we examined the efficacy of the AMX + flagellin treatment after cell depletion induced by anti-Ly6G antibody (Fig. 4). In the context of infection, more than 90 % of the lung neutrophils were eliminated by depletion as depicted in Fig. 4A-B. As expected, the combination treatment AMX + flagellin resulted in a more efficient lung bacterial clearance compared to AMX when animals received isotype control antibody (Fig. 4C). In contrast, the adjunct therapeutic effect of flagellin was abolished when neutrophils were depleted. Ultimately, these data convey that neutrophil infiltration into airways is essential for the adjunct therapeutic effectiveness of flagellin with antibiotics.

### *Flagellin treatment accelerates neutrophil migration in the lung parenchyma and changes tissue localization*

To ascertain the location of neutrophils upon lung infiltration, APC-conjugated anti-CD45 was injected intravenously before tissue sampling to label marginated and circulating leucocytes. Lung cells were next stained for flow cytometry analysis with a panel including a BV711-conjugated anti-CD45 antibody (Fig. 5A). Consequently, in infected mice untreated or treated with AMX, 50 % of lung neutrophils were marginated, whereas AMX + flagellin treatment reduced the frequency to 30 %. These results suggest that flagellin treatment accelerates the migration of neutrophils across the vascular endothelium into the lung tissue and conducting airways. A histologic assessment by hematoxylin/eosin (HE) staining and Ly6G-specific immunochemistry were next conducted on lung tissue sections (Fig. 5B-C). The infiltration and positioning of neutrophils was scored as depicted in Fig. 5D. The AMX + flagellin treatment altered the proportion of neutrophils as well as their positioning within the tissue, specifically in peripheral regions of blood vessels, bronchi, alveoli as well as within bronchi. These experiments suggest that specific distribution of neutrophils in airways is linked to the protection.
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Dynamics of lung phagocytic cells during treatment of infected mice. BALB/c mice were infected i.n. with $2 \times 10^6$ *S. pneumoniae* and treated 12 h later by i.g. administration of AMX combined with i.n. administration of flagellin (AMX + flagellin) or PBS (AMX). Lungs were collected post-treatment, and neutrophils (A, D), inflammatory $CCR2^+$ monocytes (B, E), and alveolar macrophages (C, F) were analyzed by flow cytometry. (A-C) Kinetic evolution of the number of cells ($n = 7-8$ per group) post-treatment. The data are presented as mean ± SEM. (D-F) Number of cells at 4 h post-treatment compared to mice that were left uninfected and untreated (mock) or infected but not treated ($n = 4-20$ mice per group, pool of 4 experiments). The values of individual mice and/or mean ± SEM are shown. Statistical differences between the groups were analyzed by one-way or two-way ANOVA.</figcaption></figure>

### Flagellin treatment induces an increase of neutrophil phagocytosis

We next assessed whether key neutrophil functions such as phagocytosis and ROS production were influenced by the adjunct flagellin treatment in *in vivo* and *ex vivo* experiments (Fig. 6). To investigate *in vivo* phagocytosis, pneumococcus-infected mice treated with AMX alone or AMX + flagellin were administered i.n. pHrodo-conjugated inactivated *E. coli* particles at 3 h post-treatment, and lungs were recovered 1 h later (i.e., 4 h post-treatment). The proportion of $pHrodo^+$ neutrophils (i.e., cells that have phagocytized bacterial particles) ranged from 3 to 12 % of total neutrophils (Fig. 6A-C), and there was no significant influence attributed to the treatment. However, the total number of $pHrodo^+$ neutrophils, was higher in AMX + flagellin-treated mice compared to AMX-treated group (Fig. 6B). In contrast, *in vivo* phagocytosis by macrophages and inflammatory monocytes was not altered by the treatments (Supplementary Fig. 4).

Further to this, BAL cells were recovered 4 h post-treatment and incubated *ex vivo* during 1 h with pHrodo particles (Fig. 6D-E). Neutrophil analysis demonstrated that AMX + flagellin treatment stimulates a significant increase of the pHrodo fluorescence whereas the proportion of $pHrodo^+$ cells was not affected; nearly 100 % of the neutrophils were found *ex vivo* to be phagocytosing particles (Fig. 6D). In the same, almost 100 % alveolar macrophages were phagocytic in a treatment-independent manner (Supplementary Fig. 5). Furthermore, we did not observe any significant effect of flagellin on the production of ROS by neutrophils (Supplementary Fig. 6). In conclusion, these results

indicate that neutrophil-associated phagocytosis but not ROS production correlates with the effectiveness of adjunct therapy.

### Treatments differentially regulate the airway neutrophil populations

To further elucidate the neutrophil behavior, BAL cells from animals infected and treated for 4 h with AMX or AMX + flagellin were subjected to duplex scRNA-seq to capture the whole spectrum of individual cells (Fig. 7). The DEG analysis in the entire neutrophil population indicated distinct expression profiles corresponding to each treatment (Fig. 7A). Gene enrichment analysis revealed that biological processes related to neutrophil migration, activation and phagocytosis are significantly enriched in response to AMX + flagellin treatment (Fig. 7B). On the other hand, general processes such as regulation of immune response and apoptotic signaling were predominant in AMX-treated cells. As shown in Fig. 7C-D, clustering analysis identified 7 subsets, annotated as Neutro_0 to Neutro_6, displaying distinct transcriptomic profiles, consistent with previous studies¹⁹⁻²¹. Beyond the approximate doubling of BAL neutrophils (Supplementary Fig. 3), the proportion of neutrophil subsets markedly differed between treatments (Fig. 7E). The AMX + flagellin treatment resulted in 5-to-40-fold expansion of subsets Neutro_0 and Neutro_2. Neutro_0 was characterized by the expression of genes involved in cell recruitment and migration (e.g., *Cxcl3* and *Cass4*). Neutro_2 displayed an antibacterial profile with strong expression of genes like *Retnlg*, coding for the resistin-like gamma precursor, and granule genes such as *Ngp* and *Mmp8* (Supplementary file 2). The overall
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Role of neutrophils in the efficacy of the combination treatment against pneumonia. BALB/c mice were infected i.n. with $2 \times 10^6$ S. pneumoniae. Six hours later, animals were injected with anti-Ly6G antibody or control isotype. Mice were treated 12 h post-infection by i.g. administration of AMX combined with i.n. administration of flagellin (AMX + flagellin) or PBS (AMX), or were left untreated. Lungs were collected 12 h post-treatment. (A-B) Number of neutrophils were analyzed by flow cytometry (n = 4 per group) in AMX-treated (A) and AMX + flagellin-treated animals (B). Statistical differences between the groups were analyzed by Mann and Whitney test. (C) Bacterial counts were determined by counting CFU from lung homogenates on blood agar plates (n = 7-17 per group, pool of two experiments). Statistical differences between the groups were analyzed by One-way ANOVA.</figcaption></figure>

number of Neutro_1 with anti-inflammatory and antioxidant signature (Zc3h12c and Gclm), Neutro_3, which is associated with the type I interferon response (Ifit2, Ifit3, Ifit3b and Cmpk2), and Neutro_4 was slightly altered. Finally, Neutro_6, which is marked by high expression of the metallothionein gene Mt1 was found highly specific for AMX + flagellin. Although treatments altered ratio and numbers of neutrophil subsets, they were also associated to changes in transcriptional profile. Particularly, in response to AMX + flagellin, Neutro_3 cells upregulated expression of genes involved in ROS production and phagocytosis (Fig. 7F). Lastly, although scRNA-seq identified alveolar macrophages among the BAL cells, their response to the AMX + flagellin treatment was minimal (Supplementary Fig. 7). Altogether, these findings indicate that flagellin adjunct therapy does not only increase the expansion of neutrophil specific subsets but also increases the antibacterial functions.

Discussion

In this study, we highlight the main role of airway neutrophils in the therapeutic effect of the immunity booster flagellin combined with antibiotics against pneumococcal pneumonia. High throughput analysis demonstrated that the lung immune response is associated with activation of phagocytes, i.e., neutrophils and inflammatory monocytes and their infiltration into the lungs. However, neutrophils were found to be the key players in the flagellin-mediated protection. The increased number of airway neutrophils as well as their focal localization in vascular, bronchial, and alveolar areas are the main features of adjunct therapy. Finally, scRNA-seq analysis suggested that specific airway neutrophil clusters are differentially mobilized and orientated towards recruitment, activation and antibacterial transcriptomic profiles, contributing to the flagellin treatment efficacy.

This study corroborates neutrophil number increase following flagellin treatment<sup>17,18</sup> and aims to further dissect the primary factors contributing to the efficacy. Our analyses indicated increased respiratory levels of neutrophil granule components and complement system proteins, alongside activation of antimicrobial functions in conducting airways under flagellin treatment. We also demonstrated significant regulation of genes and pathways in lungs linked to granulocyte

activation and recruitment, and phagosome maturation as evidenced by microarray. This pattern is associated with the greater phagocytic capacities of neutrophils. We hypothesized that the treatment effectiveness is not solely dependent on the number of neutrophils infiltrating the respiratory tissue, but also on the emergence of specific neutrophil subsets. The single-cell transcriptome profiling revealed that flagellin promotes neutrophil antibacterial functions. Among the seven clusters, the neutrophil subsets Neutro_0, Neutro_2, and Neutro_4, which harbor such signatures, were expanded. Flagellin-mediated reprogramming was also evident within specific clusters (such as Neutro_3), highlighting its role in boosting neutrophil antibacterial functions. The future investigations will delineate the specific contributions of each neutrophil subset to the therapeutic activity; this can be achieved by closely monitoring of key functions such as phagocytosis or ROS production.

Neutrophil heterogeneity has been demonstrated across various physiopathological contexts in both mice and humans<sup>20-23</sup>. Xie *et al.* previously identified similar neutrophil subsets, observing dynamic transitions between these subsets across various tissues and during cell activation triggered by bacterial infection, without impacting overall heterogeneity<sup>20</sup>. Among these subsets, Neutro_0 is associated with immature neutrophils, while Neutro_2 and Neutro_5 are linked to mature neutrophils, as found in the bone marrow and blood. As expected, the mature neutrophils exhibit high expression levels of genes associated with antibacterial functions. The impact of adjunct treatment on neutrophils is observed locally but there are many evidences that granulopoiesis or hematopoietic cell progenitors' outcome is altered by environmental cues, antibiotics, or flagellin<sup>20,22,24-26</sup>. It is crucial to note that neutrophil profiling focused on cells from the conducting airways. However, the tissue-associated neutrophils are altered by adjunct flagellin treatment. Spatial transcriptomic analysis could be useful as shown by Garrido-Trigo *et al.*, to unveil interaction networks involving tissue neutrophils, bacteria, and other structural and immune cells<sup>27</sup>. Indeed, further investigations are needed to define the trajectories of bone marrow- and lung-associated neutrophils.

The lung response to respiratory administration of flagellin in infected animals is comparable to previous studies in uninfected animals<sup>14,28</sup>; The dynamic is immediate and transient and is associated with
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**Fig. 5.** Flagellin treatment increases the transmigration of neutrophils into the lungs and influences neutrophil location. BALB/c mice (n = 5 per group) were infected i.n. with 2 × 10⁶ S. pneumoniae, and treated 12 h post-infection by i.g. administration of AMX combined with i.n. administration of flagellin (AMX + flagellin) or PBS (AMX), or were left untreated. Four hours post-treatment, lung neutrophils were analyzed for vascular vs parenchyma localization using anti-CD45-APC antibody injection prior to sampling followed by antibody staining for flow cytometry analysis (A) or lungs (left lobes) were analyzed by histochemistry and immunochemistry (B-D). (A) Percentage of marginated neutrophils. Statistical differences between the groups were analyzed by One-way ANOVA (*: p < 0.05). (B-D) Histological (H&E) analysis and immunodetection of neutrophils (Ly6G) in lungs of treated mice (n = 4 per group) in alveolar regions (B) and bronchi (C). (D) Neutrophil infiltration score was blindly scored using a 5-level scale, where 1 corresponds to slight, and 2 to 5 correspond to mild, moderate, marked, and severe infiltration.</figcaption></figure>

negative feedback regulation that allow to rapidly restore homeostasis. This pattern supports the concept that boosting the respiratory innate immunity through TLR5 during pneumonia does not elicit prolonged respiratory and systemic inflammation. In agreement with the histological study of Porte et al.¹⁸, the treatments did not exacerbate inflammation or damage lung architecture. Neutrophil clusters that could contribute to resolution through their antioxidant and anti-inflammatory profiles were also identified. For example, adjunct flagellin exclusively promoted the emergence of Neutro_6 subset that expresses genes with antioxidant activity, such as the methallothionein gene Mt1. It has been shown that ROS induces the production of

metallothioneins to quench ROS activity and protect against oxidative damage²⁹,³⁰. Collectively, these data suggest that modulation of inflammation and tissue damage mitigation is induced by flagellin.

In conclusion, intranasal administration of flagellin in conjunction with amoxicillin elicits a rapid and multifactorial innate immune response, involving the neutrophils as key effectors of protection. Our study suggests that targeting expansion and functional activation of specific neutrophil subsets could be a novel immunotherapeutic strategy. Furthermore, our data enrich the current knowledge on neutrophils to promote better understanding of their diversity and functions.
<figure><img src="image_11.png" /><figcaption>**Fig. 6.** Flagellin treatment increases phagocytic capacity of airway neutrophils. BALB/c mice (n = 10–12 per group) were infected i.n. with $2 \times 10^6$ *S. pneumoniae*, and treated 12 h post-infection by i.g. administration of AMX combined with i.n. administration of flagellin (AMX + flagellin) or PBS (AMX). **(A-C) in vivo assays.** At 4 h post-treatment, mice received i.n. injection of ~ $9 \times 10^6$ inactivated pHrodo *E. coli* particles in 30 µl PBS and perfused lungs were collected 1 h later. Cell suspensions were next prepared and stained for flow cytometry. Representative flow cytometry dot plot of phagocytosis of pHrodo particles (A), percentage (B) and number (C) of pHrodo⁺ neutrophils are shown. **(D-E) ex vivo assays.** Bronchoalveolar lavages were sampled 4 h post-treatment. $2 \times 10^5$ cells were incubated in microplates with $3 \times 10^6$ pHrodo-conjugated particles during 1 h at 37 °C, then labelled for flow cytometry analysis. Representative flow cytometry histogram of phagocytosis of pHrodo particles (D), and mean fluorescence intensity of pHrodo (E) in neutrophils are shown. Control corresponds to fluorescence of cells in absence of particles. Statistical differences between AMX + flagellin and AMX groups were analyzed by Mann and Whitney test (*: p < 0.05, **: p < 0.01).</figcaption></figure>

# Materials and Methods

## Bacterial strains and cultures

*Streptococcus pneumoniae* serotype 1 (clinical isolate E1586) was obtained from the National Reference Laboratory—Ministry of Health from Uruguay⁹. Bacterial cultures were done at 37 °C and 5 % CO₂ in Todd-Hewitt broth supplemented with 0.2 % yeast extract (THYB, Sigma-Aldrich, Saint-Louis, MO) or 5 % sheep blood agar plates. Working stocks were prepared as follows: THYB was inoculated with colonies from blood-agar plates and incubated until the A₆₀₀ reached 0.7–0.9 units. Cultures were stored at – 80 °C in THYB + glycerol 12 % (vol./vol.) for up to 3 months. For infection, working stocks were washed with sterile Dulbecco's Phosphate-Buffered Saline (PBS, Gibco, Grand Island, NY) and diluted in PBS to the appropriate concentration. The number of bacteria (expressed as colony forming units [CFU]) was confirmed by plating serial dilutions onto blood agar plates.

## Mouse model of infection

All animal experiments complied with current national and European regulations and ethical guidelines (animal facility agreement E59-350009, Institut Pasteur de Lille; protocols APAFIS#5164_2015 121722429127_v4 and APAFIS#16966_201805311410769_v3; GMO agreements, DUO6818 and 6820) and were conducted by qualified personnel. Animals were maintained in individually ventilated cages and were handled in a vertical laminar flow cabinet (biosafety level 2). Female BALB/cJRj mice (6–8 weeks old; Janvier Laboratories, Saint Berthevin, France) were anesthetized via intraperitoneal injection of 1.25 mg (50 mg/kg) ketamine plus 0.25 mg (10 mg/kg) xylazine in 250 µl of PBS. For infections, $2 \times 10^6$ CFU in 30 µl PBS were inoculated i.n. as described previously¹⁷,¹⁸. At selected timepoints, mice were sacrificed via the intraperitoneal injection of 5.47 mg of sodium pentobarbital in 100 µl PBS. Lungs and spleen were collected and homogenized in 1 mL PBS with an UltraTurrax homogenizer (IKA-Werke, Staufen, Germany), and viable counts were determined by plating serial dilutions onto blood agar. Bronchoalveolar lavages (BAL) fluids were obtained using three intratracheal injections of 1 ml PBS supplemented with protease
<figure><img src="image_12.png" /><figcaption>**Fig. 7.** Neutrophil subsets are differentially mobilized upon treatment of pneumococcal pneumonia. BALB/c mice (n = 4 per group) were infected i.n. with 2 × 10⁶ S. pneumoniae, and treated 12 h post-infection by i.g. administration of AMX combined with i.n. administration of flagellin (AMX + flagellin) or PBS (AMX). BAL cells were sampled 4 h after treatment and mixed at a ratio of 1/1 for duplex scRNA-seq. (A) Volcano plot identifying DEG and (B) Dot plot showing gene ontology (GO) between treatment conditions in the entire neutrophil population. (C) Uniform manifold approximation and projection (UMAP) representation of the analysis of neutrophils populations. (D) Dot plot showing scaled expression of the top 5 DEGs for each neutrophil cluster. (E) Proportions of each neutrophils subsets according to the treatment. (F) Dot plot showing GO analysis of the Neutro_3 cluster.</figcaption></figure>
inhibitors (cOmplete™, Sigma-Aldrich). Blood was sampled by retro-orbital puncture into Z-Gel micro tubes (Sarstedt, Nümbrecht, Germany) to prepare serum.

Neutrophils were depleted by intraperitoneal injection of anti-Ly6G antibody (rat IgG2b, clone NIMPR14,100 µg in 200 µL PBS), 6 h after infection of mice with S. pneumoniae. Control mice received an isotype antibody (rat IgG2b specific for HLA Bw6 clone HB-152).

## Administration of treatments

The recombinant flagellin FliC<sub>Δ174–400</sub> with a histidine tag at the C-terminus<sup>18,31</sup>, a derivative from the S. enterica serovar Typhimurium flagellin FliC, was used throughout this study. The protein FliC<sub>Δ174–400</sub> is poorly immunogenic but stimulates TLR5 signaling as assessed on reporter cell lines and in mouse models. The residual lipopolysaccharide concentration was determined to be < 20 pg per µg of flagellin. For treatment of mice, FliC<sub>Δ174–400</sub> (2.5 µg in 30 µL PBS) was administrated i.n. under light anesthesia using isoflurane inhalation (Axience, Pantin, France). Control animals received i.n. PBS alone. Amoxicillin trihydrate (AMX, Sigma-Aldrich) was prepared as a stock solution of 1.75 mg/mL in sterile water and then adjusted to a final dose of 5 µg/mouse (i.e., 0.25 mg/kg) in water before intragastric (i.e.) administration of 200 µL. Control animals received i.g. water alone.

## Quantification of gene expression and microarrays

Lung total RNA was extracted with the Nucleospin RNA II kit (Macherey Nagel, Düren, Germany) and reverse-transcribed with the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). The cDNA was amplified using SYBR-Green-based real-time PCR on a Quantstudio™ 12 K Real-Time PCR System (Thermo Fisher Scientific, Carlsbad, CA, USA). Specific primers used are listed in *Supplementary File 3*. Relative mRNA levels were determined by comparing the PCR cycle threshold (Ct) for the gene of interest to the reference gene Actb (ΔCt) and then the ΔCt values for AMX + flagellin-treated vs AMX-treated groups (ΔΔCt).

For the microarray analysis, total RNA yield and quality were assessed on the Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). One-color whole mouse (084809_D_F_20150624 slides) 60-mer oligonucleotide 8x60k v2 microarrays (Agilent Technologies) were used to analyze the gene expression. The cRNA labelling, hybridization and detection steps were carried out according to the supplier's instructions (Agilent Technologies). For each microarray, cyanine-3-labeled cRNA was synthesized from 50 ng of total RNA using the low-input QuickAmp labeling kit. RNA Spike-In was added to all tubes and used as a positive control in the labelling and amplification steps. Next, 600 ng of each purified labelled cRNA were then hybridized and washed following manufacturer's instructions. Microarrays were scanned on an Agilent G2505C scanner, and the data were extracted using Agilent Feature Extraction Software (version 10.7.3.1, Agilent Technologies). Microarray data have been deposited in the Gene Expression Omnibus (GEO) database (accession number: GSE118860). Statistical comparisons and filtering were performed with the Limma R package with 75-percentile normalization. Differentially expressed genes were considered when adjusted p-value < 0.05 after the false discovery rate had been checked with the Benjamini-Hochberg procedure. Pathways were investigated using Ingenuity Pathway Analysis software (Ingenuity Systems, Redwood City, CA, USA).

## Flow cytometry

Lungs were perfused with PBS, excised, finely minced and then digested in a solution of RPMI 1640 medium (Gibco) containing 1 mg/ml collagenase VIII and 40 µg/ml DNase I (Sigma-Aldrich) for 20 min at

37 °C. After washes, red blood cells were removed using a lysis solution (Pharmlyse, BD Bioscience). Lung cells were then suspended in a 20 % Percoll (GE Healthcare) gradient and centrifuged at 2,000 rpm without brake at room temperature for 10 min. The hematopoietic cells were recovered from the pellet and washed with PBS supplemented with 2 % fetal calf serum (FCS) and cells were filtrated before antibody labelling. BAL were sampled after 3 successive intratracheal injections of 1 ml of PBS supplemented with 5 % FCS. The BAL cells were recovered after 5 min centrifugation at 400 g. BAL and lung cells were stained with fluorescent antibodies specific for surface markers (*Supplementary File 4*) that were purchased from BD Biosciences (San Jose, CA) or BioLegend (San Diego, CA).

To detect the cells associated to lung vasculature (circulating and marginated cells), mice were intravenously injected 5 min before sampling with 5 µg of APC-labelled anti-CD45 mAb. After perfusion, lungs were removed and cell suspensions were labeled with appropriate mAbs and BV711-labeled anti-CD45 mAb to stain all leukocytes.

Data were collected on a BD LSR Fortessa and analyzed with the BD FACSDiva software. Cells were gated based on their size and granularity and propidium iodide was added before acquisition to exclude dead cells from the analysis. Neutrophils, inflammatory monocytes and alveolar macrophages were identified from viable, CD45⁺ cells (leucocytes) based on the following characteristics: macrophages (Siglec-F⁺CD11b<sup>neg</sup>CD11c⁺), neutrophils (Siglec-F<sup>neg</sup>CD11b⁺Ly6G⁺) and inflammatory monocytes (Siglec-F<sup>neg</sup>CD11b⁺Ly6C⁺CCR2⁺).

## Cytokine and chemokine production

Concentration of CCL20, CXCL2, IL-6 and TNF was determined in BAL fluids and serum by enzyme-linked immunosorbent assay (ELISA kit from eBioscience, R&D Systems or Becton Dickinson).

## Histology and immunohistochemistry

Lungs were fixed in 4 % formaldehyde. The left lobes were embedded in paraffin, and 3-to-5 µm tissue sections were stained with hematoxylin and eosin reagent or processed for Immunohistochemistry (Althisia, Troyes, France). For antigen retrieval, lung tissue sections were incubated at 68 °C for 16 h in a steamer using Target Retrieval Solution buffer (pH 9, Leica). All subsequent steps were carried out at room temperature. To block endogenous peroxidase activity, the slides were treated with 0.3 % hydrogen peroxide for 30 min. Following washes, the tissue sections were blocked with 10 % horse serum for 20 min. The slides were then incubated for 1 h with a Ly6G-specific rabbit monoclonal antibody (clone EPR22909-135, 1.5 µg/ml, Abcam) at room temperature. This was followed by a 30-minute incubation with ImmPRESS® HRP Horse Anti-Rabbit IgG Polymer (Vector Labs). The signal was visualized using DAB, and counterstaining was performed with hematoxylin. The Ly6G-stained images were blindly evaluated for neutrophil infiltration in perivascular, peribronchial, alveolar areas as well as within bronchioles on a 5-level scale, where 1 corresponded to slight, and 2 to 5 corresponded to mild, moderate, marked, and severe infiltration.

## Phagocytosis assays

Inactivated phrodo™ Red E. coli BioParticles® (Thermo Fisher Scientific) were reconstituted at a concentration of 1 mg/ml (3 × 10⁸ particles/ml) in PBS. For *ex vivo* assays, 3 × 10⁶ particles were added to 2 × 10⁵ BAL cells in complete RPMI medium in a flat bottom 96 wells plate and incubated 1 h at 37 °C. Cells were then washed with PBS, labelled with fluorescent antibodies and analysed by flow cytometry. For *in vivo* assays, 9 × 10⁶ particles in 30 µL PBS were administered i.n. under light isoflurane anesthesia 4 h after administration of the
treatments. Lung and BAL were sampled 1 h later, processed and stained for flow cytometry. Fluorescence of pHrodo particles (Excitation/ Emission maxima: 560/585 nm) was acquired on a 2020 Attune NxT Flow Cytometer (Thermo Fisher Scientific) and data were analyzed using FlowJo software (LLC, Beckton Dickinson, Franklin Lakes, NJ, USA).

Single cell RNA sequencing (scRNA-seq), demultiplexing, processing and analysis

BAL cells from 4 mice per treatment were pooled together. After cell count and viability evaluation, cell hashtag (HTO) staining (Biolegend, San Diego, CA, USA) was used following the cell-hashing protocol (TotalSeq™-A Antibodies and Cell Hashing with 10 × Single Cell 3' Reagent Kit v3.1 Protocol)³². The cells were then loaded into the Chromium controller (10 × Genomics, Pleasanton, CA, USA) and library was generated using the chromium single-cell v3.1 chemistry following manufacturer recommendations. Gene expression library was sequenced following 10 × Genomics protocol on the Illumina NovaSeq 6000 system (Illumina, San Diego, CA, USA).

Cell hashing barcodes and RNA count matrices were loaded as assays into a Seurat object³³. Matrices was first normalized using NormalizeData function with parameter normalization.method = 'CLR', and then demultiplexed using HTODemux function with default parameters. Cells either unassigned or assigned to both barcodes were discarded. Regarding RNA count matrix, cells were filtered based on the following parameters: nFeature_RNA > 100 & nCount_RNA > 500 & percent.mt < 30. Doublet cells were then discarded based on in house function derived from DoubletFinder algorithm³⁴. Resulting Seurat object was normalized and scaled using SCTransform³⁵ with method = "glmGam-Poi" and variable features.n = 8000 parameters, before applying standard Seurat workflow (RunPCA, RunUMAP, FindNeighbors and FindClusters). Most stable resolution for clustering was chosen using Clustree algorithm³⁶ and based on biological knowledge. Differentially Expressed Genes (DEGs) by clusters were calculated using PrepSCTFindMarkers and FindAllMarkers (min.pct = 0.3, only.pos = TRUE) functions and top 5 were displayed using DotPlot function to provide help for annotation. To refine the analysis, only neutrophil clusters were selected and used to run a new UMAP. The same previous functions and parameters were used to calculate DEGs between clusters or between conditions, within each neutrophil cluster or not. Volcano plot was created using EnhancedVolcano package (https://github.com/kevinblighe/EnhancedVolcano). Gene ontology enrichment analysis was performed using Metascape³⁷ with default parameters and GO Biological Processes pathways. Pathways not common to both treatment conditions were kept for the analysis. Sequencing data have been deposited at GEO (accession number: GSE269163).

Statistical analysis

Results were expressed as individual values and median [interquartile range] or mean ± standard error of the mean (SEM). Groups were compared using a Mann-Whitney test (for two independent groups), a Kruskal-Wallis one-way analysis of variance (ANOVA) with Dunn's post-test (for three or more groups), a two-way ANOVA with Sidak's multiple comparisons for time-dependent analysis of treatments. Statistical analyses were performed using GraphPad Prism software (version 8.2, GraphPad Software Inc., San Diego, CA), and the threshold for statistical significance was set to p < 0.05.

CRediT authorship contribution statement

Laura Matarazzo: Writing – review & editing, Writing – original draft, Visualization, Validation, Project administration, Methodology, Investigation. Charlotte Costa: Writing – review & editing, Writing – original draft, Validation, Project administration, Methodology,

Investigation. **Rémi Porte:** Validation, Methodology, Investigation. **Jean-Michel Saliou:** Validation, Supervision, Methodology, Investigation. **Martin Figeac:** Validation, Supervision, Methodology, Investigation. **Fabien Delahaye:** Validation, Supervision, Methodology, Investigation. **Amélie Bonnefond:** Methodology, Investigation. **Benoit Kloeckner:** Visualization, Validation, Formal analysis, Data curation. **Aymeric Silvin:** Visualization, Validation, Formal analysis, Data curation. **Florent Ginhoux:** Validation, Supervision. **Christelle Faveeuw:** Writing – review & editing, Writing – original draft, Methodology, Investigation. **Mara Baldry:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Methodology, Investigation. **Christophe Carnoy:** Writing – review & editing, Writing – original draft, Validation, Supervision, Methodology, Investigation, Conceptualization. **Jean-Claude Sirard:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

Funding

The study was funded by INSERM, Institut Pasteur de Lille, Université de Lille, the Era-Net Joint Program Initiative on Antimicrobial Resistance (ANR-15-JAMR-0001-01), the French funding body Agence Nationale de la Recherche (grant ANR-19-CE18-0030-01), and the project FAIR that received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 847786. The EGID partner was supported by the ANR Equipex (ANR-10-EQPX-07-01; 'LIGAN-MP') and France Génomique consortium (ANR-10-INBS-009).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank the UAR2014-US41-PLBS, BioImaging Center Lille - Flow Cytometry Core Facility for flow cytometry, Transcriptomics and Applied Genomics for IPA software analysis, and the PLETHA animal facility. We thank Maurane Grandmougin for proteomic analyses.

Author contributions

Contribution: LM, CCosta, MB, CF, and RP planned studies, performed experiments and analyzed data. JMS performed and analyzed the proteomic experiments. MF performed and analyzed microarray data. FD and AB performed the scRNA-seq experiments and BK, AS and FG analyzed the data. LM, CCosta, MB, CCarnoy, and JCS wrote the paper with input from all authors. JCS, MB, and CCarnoy supervised the project.

Competing interests

JCS is the inventor of the patents WO2009156405, WO2011161491, and WO2015011254 that describes the use of the recombinant flagellin of this study as an adjunct of antibiotics.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.mucimm.2024.11.009.
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