Human lung explants were obtained from adult bronchial carcinoma patients (n = 10) undergoing lung resection at local thoracic surgery centers. Peripheral tumour-free lung tissue was used which is far from bronchial tumour and was resected during the surgical intervention for lung anatomical reasons. Written informed consent was obtained from all patients. The study was approved by the ethics committee of the Charité - Universitätsmedizin Berlin, Germany (project EA2/079/13) and performed in accordance with the approved guidelines. The healthy tissue was edited into small pieces (weight app. 0.1-0.2 g/piece) and cultivated in RPMI 1640 medium (RPMI) supplemented with or without 10% fetal calf serum (FCS; Merck, Darmstadt, Germany) at 37°C and 5% CO₂, as described before (Fatykhova et al., 2015; Peter et al., 2017).

Motile and leached-out cells of human lung tissue explants were also used for infection studies. This cell suspension was obtained from the bottoms of sample containers and comprised mostly erythrocytes, lymphocytes and primary alveolar macrophages. Erythrocytes were lysed as described by Vuorte et al. (Vuorte et al., 2001). Briefly, the cell suspension was pelleted and resuspended in H₂O for 15 sec. PBS was added, samples were centrifuged and the cell pellet was adjusted to 10⁵ cells/mL and seeded into 24-well plates. After over-night incubation cells were challenged with Francisella as described below.

Alveolar macrophages (AM) were isolated from human lung tissue as described above (Berg et al., 2017). Briefly, human lung tissue was repeatedly perfused with Hanks’ balanced salt solution (HBSS), and AM were seeded on glass coverslips in 12-well plates, 1x10⁵ cells/well in RPMI medium. After 4 h of adherence (37°C, 5% CO₂) remaining erythrocytes were removed by repeated washing with HBSS. AM were cultured in RPMI medium supplemented with 2% FCS for 2 days.

The human macrophage-like cell line U937 (ATCC CRL-1593.2) and leached-out cells of human lung tissue (see above) were cultivated in RPMI medium supplemented with 10% FCS at 37°C and 5% CO₂. Prior to the infection assays U937 cells were stimulated with PMA (phorbol-12-myristate-13-acetate, 1 mg/mL in dH₂O [Sigma-Aldrich Chemie]) at a concentration of 1:20,000 for 36 h.

Strains used in this study are listed in Table 1 . Francisella strains were cultivated in medium T (MT; (Pavlovich and Mishan'kin, 1987; Becker et al., 2016)) or on MT agar plates supplemented with hemoglobin and charcoal (MTKH plates, (Tlapak et al., 2018)).

Human lung tissue explants were inoculated with 10⁶ CFU overnight grown Francisella strains for 2 h. Explants were washed three times with 2 mL plain RPMI and treated with 50 µg/mL gentamicin for 1 h to eliminate remaining extracellular bacteria. After washing them three-times and applying fresh plain RPMI, the lung explants were incubated up to 72 h. To determine the CFU/mL at various time points of the infection, human lung tissue was homogenized with Lysing Matrix D (MP Biomedicals) and saponin (0.001%; Sigma-Aldrich Chemie) in FastPrep-24 (MP Biomedicals) for 20 sec at 4 m/s and serial dilutions were plated on MTKH agar. Culture supernatants were collected and stored at -80°C until further analysis (see below).

For co-infection of Fth LVS and Fth A-660, human lung tissue explants were simultaneously challenged with both strains for 2 h (10⁶ (LVS) and 10⁵ (A-660) bacteria per explant). After washing and gentamicin treatment (same procedure as above), explants were homogenized and suspension was plated onto MTKH agar plates, half of which were supplemented with erythromycin (Ery), respectively, to distinguish between the two Fth strains. Belonging to biovar II Fth LVS is erythromycin-resistant, whereas Fth A-660, as a biovar I strain, is erythromycin-sensitive. Therefore, only Fth LVS grows on MTKH agar supplemented with Ery (MTKH+Ery; constituting Fth LVS CFU/mL). Fth A-660 CFU/mL values were calculated by subtracting CFU/mL gained from MTKH+Ery plates from total CFU/mL rates received on MTKH plates (on which both strains are able to grow).

For infection of human macrophages (AM, U937), cells were seeded at a concentration of 5 × 10⁵ cells/mL and challenged with overnight grown Francisella strains for 2 h (multiplicity of infection (MOI) of 10). After removing the bacterial suspension, cells were washed and treated with 50 µg/mL gentamicin for 1 h. Subsequently, plain medium was added and cells were incubated up to 72 h. To determine the CFU at various time points of infection, cells were lysed with saponin (0.001%), and serial dilutions were plated on MTKH agar.

For the co-infection assay, U937 cells were seeded at a concentration of 5 × 10⁵ cells/mL and infected with a mixture of Fth LVS and Fth A-660 bacteria (1:1; in a total MOI of 10) for 2 h. Cells were treated with 50 µg/mL gentamicin for 1 h, afterwards washed and incubated up to 72 h (same procedure as for infection of human macrophages, see above). At various time points, U937 cells were lysed with 0.001% saponin, and bacterial suspension was plated on MTKH agar plates, half of which were supplemented with erythromycin (Ery), respectively, to distinguish between the two Fth strains as described above.

Isolated AM were challenged with Fth A-660 as described in section “Infection of human lung tissue and cells”. After 48 h AM were fixed as described before (Berg et al., 2017) and stained with an established cell marker for AM CD68 (abcam, Cambridge, UK) and anti-Ft LPS-FITC antibody (F11FITC, (Grunow et al., 2000)). Immunofluorescence of AM was analyzed using a LSM 780 [(objectives: Plan Apochromat 63x/1.40 oil DIC M27 and 40x/1.30 oil DIC M27), Carl-Zeiss, Jena, Germany].

For immunohistochemistry of infected human lung tissue, explants were challenged with 10⁶ bacteria/mL for 24 h and 48 h. Afterwards the samples were fixed in 3% paraformaldehyde for 48 h, embedded in paraffin and routinely processed for histology and immunofluorescence staining as described before (Peter et al., 2017; Hönzke et al., 2022). For characterization of infected cells, the specific cell marker CD68 for AM and anti-Ft LPS antibody (Grunow et al., 2000) were used, followed by incubation with corresponding secondary antibodies. Nuclei were subsequently counterstained with DAPI (Sigma Aldrich). Immunofluorescence of human lung slices was analyzed by spectral confocal microscopy using a LSM 780 [(objectives: Plan Apochromat 63x/1.40 oil DIC M27 and Plan Apochromat 40x/1.40 oil DIC M27), Carl-Zeiss, Jena, Germany]. Based on a spectral image lambda stack, linear unmixing of tissue autofluorescence and overlapping spectra of fluorochromes were performed using ZEN 2012 software (Carl-Zeiss, Jena, Germany). To reveal lung and cell morphology, images were combined with Differential Interference Contrast (DIC). All image sets were acquired using optimal configuration regarding resolution and signal to noise ratio. Images were processed using ZEN 2012.

Human lung explants were challenged with 10⁸ Francisella bacteria per mL for up to 48 h. Electron microscopy of human lung explants was done essentially as described before (Weinheimer et al., 2012). Lung tissue was fixed by immersion in 4% Formaldehyde, 2.5% glutaraldehyde (in 50 mMHepes-buffer) and post fixed with 1% OsO4 (1 h), 0.1% tannic acid (in 50 mM Hepes buffer, 30 min) and 2% uranyl acetate (2 h). Samples were dehydrated in ethanol and embedded in epon resin. Thin sections were produced using an ultramicrotome (UC7, Leica, Wetzlar, Germany) and stained with 2% uranyl acetate (20 min) followed by lead citrate (2 min). Sections were examined using a transmission electron microscope (Tecnai Spirit, Thermo Fisher/FEI) operated at 120 kV. Images were recorded using a CCD-camera (Phurona, Emsis, Münster, Germany).

Supernatants of infected human lung tissue explants were collected 24 h, 48 h and 72 h post infection. After centrifugation (10 min, 5000 g, 4°C) 100 µl aliquots were stored at liquid nitrogen until further investigations. Samples were sterilized using 0.2 µm filter (Sartorius, Göttingen, Germany) prior to measurement of cytokines and chemokines. Cytokines and chemokines were analyzed in cell culture supernatants using the LegendPlex assay (BioLegend, USA) according to the manufacturer’s instructions. For the analyzed biomarkers, the detection limits of the LegendPlex assay were, as follows: granulocyte colony-stimulating factor (G-CSF, 33.66 pg/mL), interferon-α (IFNα; 3.11 pg/mL), IFNγ (3.35 pg/mL), interleukin (IL) 1ß (4.23 pg/mL), IL-2 (1.93 pg/mL), IL-4 (2.22 pg/mL), IL-5 (2.7 pg/mL), IL-6 (3.09 pg/mL), IL-8 (2.53 pg/mL), IL-9 (3.12 pg/mL), IL-10 (1.9 pg/mL), IL-12p70 (4.75 pg/mL), IL-13 (3.93 pg/mL), IL-17A (3.39 pg/mL), IL-17F (2.07 pg/mL), IL-21 (4.04 pg/mL), IL-22 (2.16 pg/mL), interferon-γ–induced protein-10 (IP-10 = C-X-C motif chemokine ligand 10 (CXCL-10), 4.48 pg/mL), monocyte chemotactic protein-1 (MCP-1 = CC-chemokine ligand 2 (CCL-2); 6.04 pg/mL), tumor necrosis factor-α (TNFα, 2.41 pg/mL) and vascular endothelial growth factor (VEGF, 31.41 pg/mL).

Statistical analysis was performed with GraphPad Prism 9 software. For comparison between the two analyzed groups (LVS vs. A-660), a two-tailed test t was used. For comparison of three groups (LVS vs. A-660 and A-660 10⁵, respectively), one-way ANOVA and Kruskral-Wallis test with multiple comparison were performed.

In order to determine the growth of intracellular Francisella, represented by the colony forming units (CFU), the human lung explants needed to be homogenized and lysed after infection. To test if Francisella is able to survive the homogenization, 10⁵ Fth LVS bacteria were treated in PBS supplemented with 0.001% saponin for 20 sec with 4 m/s using a FastPrep homogenizer. As shown in Figure S1A , the homogenization with FastPrep did not significantly reduce the bacterial load of the LVS compared to untreated and vortexed samples. In order to monitor a possible extracellular replication of Francisella, we used Fth LVS ΔiglC in all infection assays as a control strain. This mutant strain is unable to escape the phagosome and is therefore, unable to replicate intracellularly (Golovliov et al., 1997; Golovliov et al., 2003; Lindgren et al., 2004; Santic et al., 2005). When U937 macrophages were infected with LVS and LVS ΔiglC for 2 h, washed and incubated for 24 h and 48 h, respectively, CFU/mL of both, LVS and LVS ΔiglC, increased over time ( Figure S1B ), demonstrating that Fth replicates in co-culture with U937 cells. Treatment by gentamicin inhibited the extracellular replication ( Figure S1B ). After observing a minimal increase of bacteria (OD_600nm) cultivated in RPMI + 10% FCS (data not shown), for all following infection assays, RPMI medium was used without FCS to minimize a possible extracellular replication of Fth in our model.

The experimental procedure of the ex vivo infection model was adapted for Francisella, as follows ( Figure 1 ): 10⁶ Francisella bacteria were injected into human lung tissue explant portioned into three punctures (~ 33 µl each). The infection was performed at 37°C and 5% CO₂ for 2 h. Medium was completely removed; lung explants were thoroughly washed and treated with 50 µg/mL gentamicin for 1 h to eliminate the remaining extracellular bacteria. Subsequently, medium was removed, followed by a complete rinsing of explants. Afterwards, fresh plain RMPI medium was added and the lung explants were incubated for up to 72 h. The human lung tissue was further used (1) to determine the intracellular growth and (2) to identify the infected cell types. To achieve this, (1) human lung explants were homogenized, lysed and plated on agar plates to determine the CFU per g lung tissue ( Figure 1 ). (2) The analysis by differential interference contrast and electronic microscopy was conducted after fixing the human lung explants and staining them with antibodies for Fth as well as selected cell markers.

In this study we used Fth LVS, Fth LVS ΔiglC and a human isolate Fth (Fth A-660) obtained from a tularemia patient exhibiting pneumonia (Appelt et al., 2019), as well as an environmental Francisella species (Francisella sp. strain W12-1067, F-W12) isolated from a cooling tower in Germany (Rydzewski et al., 2014). It is yet not known if the environmental species F-W12 is pathogenic for humans, but the species possesses some well-known virulence factors of Francisella and is able to persist in mouse macrophages and Acanthamoeba lenticulata (Rydzewski et al., 2014; Köppen et al., 2019). By using the human lung ex vivo infection model, an increase of CFU/g could be shown in the lung explants over 72 h for Fth LVS and Fth wild-type A-660. During this process, it became obvious that the human isolate Fth A-660 replicated to a greater extent than Fth LVS leading to a higher CFU count per g lung tissue ( Figure 2A ). Over a time period of 72 h, a 22-fold CFU-increase/g tissue was observed for Fth LVS and a 49-fold increase for A-660 (A-600: 2.7 × 10⁷ CFU/g; LVS: 2.4 × 10⁵ CFU/g; p = 0.0047). A similar trend was obtained when a lower bacterial load of Fth A-660 was used (10⁵ bacteria instead of 10⁶ per lung explant). Here, we observed an 86-fold CFU-induction/g lung tissue (A-660 10⁵ after 72 h: 5 × 10⁶ CFU/g, LVS: 2.4 × 10⁵ CFU/g; p = 0.3537). In contrast, the bacterial load of Fth LVS ΔiglC did not increase in lung explants over time, instead a minor reduction was obtained (0.56-fold induction from 24 h to 72 h, Figure 2A ). Neither replicated the environmental Francisella strain F-W12 in the human lung explants, but the strain persisted more or less stable over a time period of 72 h (1.6-fold induction; Figure 2A ). Hence, the human lung ex vivo infection model confirmed the theoretically expected intracellular growth of different Francisella strains. To further underpin the reliability of the observed results, five other Fth isolates (one animal isolate obtained from a Eurasian beaver: A-271; four human isolates obtained from tularemia patients: A-663, A-820, A-981, A-1308) were investigated. All tested isolates showed an increase of CFU per g lung tissue comparable to those observed for Fth A-660 after 48 h ( Figure 2B ).

We further aimed to identify the cell types involved in the course of a Francisella infection of the human lung by applying spectral immunofluorescence to Fth A-660-infected lung explants. After 48 h, parts of highly infected areas were found. Especially, the assumed injection spot showed a high concentration of Fth A-660 bacteria, as shown in Figure 3A . Apart from the injection sites, Fth A-660 was primarily detected in human alveolar macrophages in distinct areas (see Figure 3B ). These results were confirmed by examining Fth A-660 infected lung explants by electron microscopy ( Figure 3C ). However, Fth A-660 was not only detected in alveolar macrophages, but also sporadically in other cell types, including lymphocytes, granulocytes and fibrocytes, as well as, extracellular and within the connective lung tissue ( Supplemental Figure S2 ).

As a next step, we assessed how Francisella activated the host response in ex vivo infected human lungs by quantifying the induced cytokine and chemokine profile. This was performed by collecting and analyzing the culture supernatants of lung explants infected by Fth LVS, Fth LVS ΔiglC, Fth A-660 and F-W12 24 h, 48 h and 72 h post infection to determine the levels of cytokines and chemokines (e.g. IL-1β, IL-6, TNF-α, IL-8, G-CSF, MCP-1, IP-10 and VEGF). Just as the individual immune response differs from human to human, the induced cytokine and chemokine production varied between ex vivo infected lung explants (see Figure S2 ) and remained not to be statistically significant. Nevertheless, the following tendencies could be observed: The cytokine and chemokine response differed between Fth A-660 and Fth LVS. Fth A-660 seemed to induce a lower secretion of IL-1β (p = 0.0519 after 48 h), MCP-1 and IL-6 compared to Fth LVS ( Figure 4 ). After 24h, a lower concentration of IP-10 seemed to be induced by A-660, but after 72 h, a higher concentration of IP-10 was detected in A-660-infected human lung explant supernatants compared to LVS ( Figure 4 ). We did not identify distinctive differences in secretion of IL-8, G-CSF and VEGF between Fth LVS and Fth A-660 infected lung explants ( Figure S2 ). In general, the environmental F-W12 strain seemed to induce higher levels of cytokines, including TNF-α, in the infected lung explants ( Figures 4 , S2 ).Various further chemokines and cytokines, including IL-2, IL-4, IL-5, IL-9, IL-10, IL-12p70, IL-13, IL-17A, IL-17F, IL-21, IL-22, IFN-α and IFN-γ, were investigated but remained below the detection limits (data not shown).

We next aimed to compare the results obtained by the ex vivo model with “classical” single cell type infection models using U937 macrophages and primarily isolated human alveolar macrophages as well as multicellular primarily isolated mobile human lung cells ( Figures 5A–C ). Cells were infected with Fth LVS, Fth LVS ΔiglC, Fth wild-type A-660 and F-W12 (MOI = 10) for 2 h and subsequently treated with gentamicin (50 µg/mL for 1 h) to eliminate remaining extracellular bacteria. After 3 h, 24 h, 48 h and 72 h of infection the CFU/mL was determined. Here, as observed in the human lung infection model, only Fth LVS and Fth A-660 were able to replicate within cells tested ( Figures 5A–C ). In U937 macrophages, both strains similarly replicated ( Figure 5A ), whereas in primary alveolar macrophages ( Figure 5B ) and primary mobile lung cells ( Figure 5C ), the Fth LVS strain showed higher growth rates compared to Fth A-660 (6-fold higher in alveolar macrophages and 19-fold in mobile lung cells, Figures 5C ). Hence, these findings differ from the results obtained using the human lung ex vivo infection model, in which Fth A-660 showed a higher replication rate compared to Fth LVS ( Figure 2A ). However, Fth LVS ΔiglC and F-W12 did not significantly replicate in these cell culture models, except for an increase of CFU/mL observed for ΔiglC in infected primary alveolar macrophages after 72 h, indicating extracellular growth (64-fold induction, Figure 3B ). Moreover, the intracellular localization of Fth A-660 in primarily isolated alveolar macrophages was confirmed by spectral immunofluorescence, as shown in Figure 5D .

Having observed differences in intracellular growth of Fth LVS and Fth A-660 depending on the infection model used, ex vivo (lung explants) or in vitro (U937), we tested both models by co-infection assays. U937 cells were simultaneously infected with both strains at a ratio of 1:1 representing a MOI of 10 in total. Cells were infected for 2 h and subsequently treated with gentamicin for 1 h. After 3 h, 24 h, 48 h and 72 h of infection, the CFU/mL was determined by lysing cells and plating onto MTKH agar plates partly supplemented with erythromycin to distinguish the two strains. Fth LVS belongs to erythromycin-resistant biovar II, whereas Fth A-660 belongs to erythromycin-sensitive biovar I. In Figure 6A , the CFU percentage of Fth LVS and Fth A-660 per 5 × 10⁵ U937 cells is shown. After infection and gentamicin treatment (3 h), LVS represented 59.9% and A-660 40.1% of bacteria obtained intracellularly in U937 macrophages (p = 0.002). In the course of infection, the percentage of LVS continuously increased up to 93.2% after 72 h (p < 0.000001). Thus, LVS significantly outcompeted A-660 during a co-infection in U937 macrophages. In contrast, the A-660 wild-type strain outcompeted the LVS strain during a co-infection of human lung explants by both strains ( Figure 6B ). Human lung explants were equally infected with both strains after 3 h (LVS: 50.8%; A-660: 49.2%). After 24 h of co-infection, LVS represented a significantly higher percentage in the explants (69.1%, p = 0.0029), whereas the proportion of A-660 continuously increased up to 73.5% after 48 h and 72 h (72h: p = 0.0231). Hence, the attenuated phenotype (reduced virulence) of Fth LVS is only detectable in co-infections using human lung explants.