Trimodulin supports antibacterial defence and restricts inflammation in preclinical pneumonia models

Abstract

Background: Severe community-acquired pneumonia remains a global health challenge with high mortality despite advances in antibiotic therapy and supportive care. Immunoglobulin therapies, especially IgM-containing ones, have shown promise in enhancing host defence and reducing inflammation. The CIGMA trial highlighted the potential of trimodulin to lower mortality in patients with severe community-acquired pneumonia with high C-reactive protein and low IgM levels.

Methods: We investigated the protective effects of trimodulin on clinical status, bacterial burden, lung integrity and inflammatory responses in murine models of lung injury, including both ventilator-induced lung injury and infection-induced models with nonsterile inflammation.

Results: In mice, trimodulin significantly protected against lethal pneumococcal pneumonia by reducing bacterial burden and disease severity while preserving alveolar barrier integrity and limiting lung oedema. The antibacterial action of trimodulin was mediated through opsonophagocytosis, and its anti-inflammatory effects operated independently of the latter. When combined with ampicillin, trimodulin exhibited enhanced suppression of inflammation.

Conclusion: Our findings in preclinical pneumonia models suggest that trimodulin could be a promising therapy for severe community-acquired pneumonia. We provide evidence that trimodulin enhances host defence, reduces detrimental pulmonary inflammation and barrier dysfunction, and limits pulmonary oedema, which may explain the beneficial effects observed in patients with severe community-acquired pneumonia.

Overview of the study. CAP: community-acquired pneumonia; ctr: control; hpi: hours post infection; HVT: high tidal volume ventilation; i.n.: intranasal; i.p.: intraperitoneal; i.v.: intravenous; NV: non-ventilated; OD: optical density; OPKA: opsonophagocytosis killing assay; sCAP: severe community-acquired pneumonia; S. pneumoniae: Streptococcus pneumoniae; VILI: ventilator-induced lung injury.

FIGURE 1

Trimodulin alleviated pneumococcal pneumonia burden and reduced lung oedema. a) Preventive trimodulin treatment scheme in murine pneumococcal pneumonia. B6J mice were intravenously (i.v.) treated with trimodulin (7.7 µL·g⁻¹=384 mg·kg⁻¹ body mass) or buffer control 2 h prior to (−2 h post infection (hpi)) and 22 hpi S. pneumoniae serotype 3 or PBS mock infection. b) Body weight (% of start), c) rectal body temperature and d) clinical score (supplementary table S2) over time. Data are presented as curves with mean±sem, n=12. Statistical significance determined with mixed-effects models (restricted maximum likelihood) and Šídák's multiple comparisons test between infected groups. Time point 72 hpi not represented by buffer control (ctr) + S. pneumoniae-infected group and therefore excluded from the statistical analysis. e) Per cent survival using Kaplan–Maier curves. A log-rank (Mantel–Cox) test was done between infected groups. f) Aspartate aminotransferase (AST) and g) blood urea levels in plasma of mice, measured by Cobas 8000 C701 (Roche Diagnostics). Statistical significance determined using one-way ANOVA with Šídák's multiple comparisons test between PBS and S. pneumoniae-infected groups, n=0–6. h) Bronchoalveolar lavage fluid (BALF) samples from different time points were analysed for protein content as a representation of lung barrier function. Statistical significance determined using two-way ANOVA with Tukey's multiple comparisons test. i, j) Haematoxylin and eosin-stained lungs sections were analysed and scored by a pathologist for i) alveolar oedema and j) perivascular oedema. ctr: control; i.n.: intranasal. *: statistical difference between buffer ctr + S. pneumoniae versus trimodulin + S. pneumoniae groups; ^#: statistical difference between buffer ctr + mock PBS versus buffer ctr + S. pneumoniae groups; ^¶: statistical difference between trimodulin + mock PBS versus S. pneumoniae-infected groups. *: p<0.5; **^/##/¶¶: p<0.01; ***^/###/¶¶¶: p<0.001; ****^/####: p<0.0001.

FIGURE 2

Trimodulin reduces bacterial burden. a) Prophylactic treatment scheme in murine pneumococcal pneumonia. B6J mice were treated intravenously (i.v.) with trimodulin (7.7 µL·g⁻¹=384 mg·kg⁻¹ body mass) or buffer control 2 h prior to (−2 h post infection (hpi)) and 22 hpi with S. pneumoniae serotype 3 or PBS mock infection. b–e) Bacterial burden in b) bronchoalveolar lavage (BAL), c) lungs, d) blood and e) spleen. CFU data were log transformed and data are presented as mean±sem, n=12. Statistical significance determined using two-way ANOVA with Šídák's multiple comparisons test. Dotted lines indicate limit of detection. f–i) Lung sections were immunohistochemically stained for pneumococci and the presence of bacteria was scored in four lung compartments: f) alveolar parenchyma, g) perivascular space, h) subpleural space and i) mediastinal adipose tissue. Scores presented as mean±sem, n=0–6. j–m) Corresponding representative images of infected, buffer control-treated (top row) and infected trimodulin-treated mice (bottom row) at 48 hpi of the j) alveolar parenchyma, k) perivascular space, l) subpleural and m) mediastinal adipose tissue. Free streptococci (red) marked with solid ovals. Phagocytosed streptococci (red) within neutrophils marked with small, solid circles or within macrophages marked with large, dotted circles. Original magnification ×600, scale bar: 100 µm. ctr: control; i.n.: intranasal; S.pn.: S. pneumoniae. ****: p<0.0001.

FIGURE 3

Trimodulin translocates into alveolar spaces in vivo and eliminates S. pneumoniae via opsonophagocytosis in vitro. a) Preventive trimodulin treatment scheme in murine pneumococcal pneumonia. B6J mice were intravenously (i.v.) treated with trimodulin (7.7 µL·g⁻¹=384 mg·kg⁻¹ body mass) or buffer control 2 h prior to (−2 h post infection (hpi)) and 22 hpi with S. pneumoniae serotype 3 or PBS mock infection. b–d) Levels of human b) IgM, c) IgA and d) IgG levels in bronchoalveolar lavage fluid (BALF) of experimental mice. e–g) Levels of human e) IgM, f) IgA and g) IgG levels in plasma of experimental mice. Ig levels were determined by semi-quantitative ELISA measurements. For b–g, data presented as mean±sem. Statistical significance determined by two-way ANOVA with Šídák's multiple comparisons test between trimodulin-treated groups, n=10–13. h) Growth curves of different S. pneumoniae serotypes (STs) in liquid media in the presence of buffer (negative ctr), 5 mg·mL⁻¹ trimodulin or 3 µg·mL⁻¹ ampicillin (positive ctr). Optical density at 600 nm (OD₆₀₀) was measured every 30 min. Three independent experiments were performed in triplicate. Representative graph shown with data presented as mean±sem, n=3. i) Opsonophagocytic killing assay of buffer ctr, trimodulin or World Health Organization (WHO) anti-streptococcus sera (pneumococcal vaccinated donors), using neutrophil-like HL60 cells as phagocytes, and live S. pneumoniae as target cells. Four independent experiments were performed in triplicate. Data are presented as mean±sem, n=4. Statistical significance determined by two-way ANOVA with Tukey's multiple comparisons test. There was no significant difference between trimodulin and WHO (positive ctr). ctr: control; i.n.: intranasal; S.pn.: S. pneumoniae. ^#: trimodulin versus buffer ctr; ^¶: WHO versus buffer ctr; *: p<0.5; **^/##: p<0.01; ***^/¶¶¶: p<0.001; ****: p<0.0001.

FIGURE 4

Trimodulin and ampicillin therapy alone and in combination reduced bacterial burden. a) Trimodulin therapeutic treatment scheme in murine severe community-acquired pneumonia models. Mice were intranasally (i.n.) infected with a dose of 5×10⁶ S. pneumoniae serotype 3 or PBS mock infection. At 22 h post infection (hpi), mice received treatment according to the group assignments (384 mg·kg⁻¹ body mass trimodulin or buffer control (ctr) intravenously (i.v.) and 0.4 mg per mouse of ampicillin or NaCl ctr intraperitoneally (i.p.)). At 24 hpi, corresponding groups underwent ventilation for 6 h with a tidal volume of 12 mL·kg⁻¹, a positive end-expiration pressure of 2 cmH₂O and an inspiratory oxygen fraction of 75%, or remained unventilated (NV ctr). All groups were analysed at 30 hpi. b) Peak inspiratory pressure (PIP) was measured every 10 min in mice receiving mechanical ventilation. Data are presented as the mean. c) Body temperature of NV ctr mice was assessed as a disease parameter at 30 hpi. Data presented as mean±sem. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test. Significant differences are reported only for the placebo (buffer ctr and NaCl ctr) groups when comparing PBS-treated mice with S. pneumoniae-infected mice. If applicable, significant differences among various S. pneumoniae experimental groups are provided for all group comparisons. d–g) Bacterial burden was determined at 30 hpi in all experimental groups in d) bronchoalveolar lavage (BAL), e) lungs, f) blood and g) spleen. Data presented as mean±sem. Statistical significance was determined using two-way ANOVA with Šídák's multiple comparisons test, tested between NV ctr and ventilated groups, n=6–12. ctr: control; i.n.: intranasal; S.pn.: S. pneumoniae. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

FIGURE 5

Combined trimodulin and ampicillin therapy reduced pulmonary and systemic inflammation. a) Trimodulin therapeutic treatment scheme in murine severe community-acquired pneumonia models. Mice were intranasally (i.n.) infected with a dose of 5×10⁶ S. pneumoniae serotype 3 or PBS mock infection. At 22 h post infection (hpi), mice received treatment according to the group assignments (384 mg·kg⁻¹ body mass trimodulin or buffer control (ctr) intravenously (i.v.) and 0.4 mg ampicillin or NaCl ctr). At 24 hpi, corresponding groups underwent ventilation for 6 h with a tidal volume of 12 mL·kg⁻¹, a positive end-expiration pressure of 2 cmH₂O and an inspiratory oxygen fraction 75%, or remained unventilated (NV ctr). All groups were analysed at 30 hpi. b) Degree of pneumonia modified from Li Bassi et al. [26]. Grading ranged from 0 (no pneumonia) to 1 (minimal), 2 (mild), 3 (moderate), 4 (severe) or 5 (massive). c) Acute lung injury (ALI) score by Matute-Bello et al. [27]. d) Lung area affected (%). Data presented in b–d as mean±sem, n=1–4. e) Scanned images of cross sections of lungs stained with haematoxylin and eosin. Scale bar: 4 mm. f–i) Cytokine measurements by multiplex ELISA in plasma. Data presented as mean±sem, n=5–8. Statistical significance determined by two-way ANOVA with Dunnett's multiple comparison test, tested against placebo (buffer ctr and NaCl ctr) versus S. pneumoniae-infected groups. ctr: control; S.pn.: S. pneumoniae. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

FIGURE 6

Trimodulin and ampicillin combination therapy reduced inflammatory gene expression upon S. pneumoniae infection. a) Trimodulin therapeutic treatment scheme in a murine severe community-acquired pneumonia model. Mice were infected with a dose of 5×10⁶ S. pneumoniae serotype 3. At 22 h post infection (hpi), mice received treatment according to group assignments (384 mg·kg⁻¹ body mass trimodulin or buffer control (ctr) intravenously (i.v.) and 0.4 mg ampicillin or NaCl ctr). All groups were analysed at 30 hpi. b) Volcano plot displaying differentially expressed genes (DEGs) between trimodulin and ampicillin combination therapy versus placebo (buffer ctr and NaCl ctr) treatment. DEGs with increased (log₂ fold changes >0.5 and adjusted p<0.05) or decreased expression (log₂ fold changes <−0.5 and adjusted p<0.05) are denoted in red and blue, respectively. c) Hallmark gene sets of DEGs in the indicated experimental comparisons. In the case of trimodulin alone, no enriched gene sets were detected. When using the combination therapy of ampicillin and trimodulin, lower adjusted p-values and a higher number of regulated genes were enriched compared to ampicillin therapy alone. d) Dot plot of log₂ fold changes for different comparisons for the selected key genes, which belong to the gene sets “complement activation” and “inflammatory response”. Differential expression analyses were performed with DESeq2 and the Wald test was used to identify DEGs. p-values were adjusted for multiple testing using the Benjamini–Hochberg method. Pathway enrichment analyses were performed using the R package tmod (www.r-project.org). S.pn.: S. pneumoniae; Tnfɑ: tumour necrosis factor ɑ; NfκB: nuclear factor κB; Ifn: interferon.

FIGURE 7

Trimodulin diminished ventilator-induced lung injury. a) Preventive treatment scheme in murine ventilator-induced lung injury model. Mice were treated intravenously (i.v.) with trimodulin (384 mg·kg⁻¹ body mass) or buffer control (ctr) 30 min prior to ventilation start. b) Mean arterial pressure (MAP), c) heart rate (HR) and d) oxygen saturation (S_pO2) measured at 10-min intervals during 240 min of high tidal volume (HVT) ventilation. Statistical significance was determined using two-way ANOVA. e–i) Blood gas parameters e) pH, f) bicarbonate (HCO₃⁻), g) standardised arterial oxygen tension (stP_aO2), h) arterial carbon dioxide tension (P_aCO2) and i) standard base excess (SBE) in arterial blood measured after no ventilation (NV) or 240 min of HVT ventilation. Statistical significance determined by two-way ANOVA with Šídák's multiple comparisons test for selected comparisons (between treatments and between ventilation regimes). j) Airway opening pressure (AOP), k) compliance (C) and l) elastance (E) normalised to 1 and measured at 5-min intervals during 240 min of HVT ventilation, analysed using mixed-effect modelling (restricted maximum likelihood). Data presented as mean±sem, n<15. m) Mouse lung permeability measured as the ratio of mouse albumin concentration in plasma and bronchoalveolar lavage fluid (BALF) samples. n) Proportions and numbers of BALF neutrophils (Ly6G⁺CD11b^hi) measured by flow cytometry. Statistical significance determined in m and n by two-way ANOVA with Šídák's multiple comparisons test for selected comparisons (between treatments and between ventilation regimes). *: p<0.05; **: p<0.01; ****: p<0.0001.