C3HeB/FeJ mice with chronic Mycobacterium avium complex pulmonary infection exhibit impaired respiratory function but not necrotising granulomatous disease

Abstract

Background: Mycobacterium avium complex (MAC) is driving a global rise in pulmonary disease (MAC-PD) characterised by chronic infection, granulomatous inflammation and impaired respiratory function. Better animal models are needed to screen candidate therapies targeting bacteria and immune-mediated tissue injury. The C3HeB/FeJ mouse was previously reported to model necrotic granulomatous lung infection in MAC-PD following infection with a low-dose inoculum of the clinical isolate MAC2285R. We investigated whether this model was reproducible with variations in MAC strain and inoculating dose.

Methods: Six-week-old female C3HeB/FeJ mice were infected intratracheally with a clinical isolate of MAC (MAC2285R) or reference strains (MAC104 or MAC101). Mice were culled at 4-weekly intervals post-infection until week 12. Lungs, spleen and liver were harvested for bacterial burden enumeration and histological examination. Whole body plethysmography (WBP) was performed weekly to measure changes in respiratory function (Buxco system).

Results: C3HeB/FeJ mice infected with low dose inoculum of MAC2285R infection exhibited increasing bacterial lung infection for 8 weeks (p < 0.05), followed by stable lung burden from weeks 8-12. High dose inoculum resulted in stable lung bacterial burden over 12 weeks. Histological analysis revealed only mild inflammatory changes in both low and high dose inoculum groups at weeks 4, 8 and 12 post-infection, with no evidence of necrotising or non-necrotising granulomatous inflammation. Surrogate measures of respiratory effort (frequency, tidal volume, inspiratory and expiratory flow rates) were increased in mice with high dose inoculum compared to uninfected controls (p < 0.001), but not low dose inoculum. Similar findings on lung bacterial burden and histological analysis were found in mice infected with low- and high-dose inoculum of MAC104 and MAC101. MAC104 infection caused greater changes in respiratory function, whereas MAC101 did not significantly affect breathing patterns.

Conclusion: The C3HeB/FeJ mouse is susceptible to chronic MAC infection from intratracheal infection with reference and clinical isolates, but this was not associated with severe granulomatous inflammation as previously reported. A low dose inoculum generated a proliferative lung infection, whereas high dose inoculum resulted in chronic, stable lung bacterial burden. Mice with high-dose inoculum MAC2285R and MAC104 infection also displayed evidence of increased respiratory effort.

Supplementary information: The online version contains supplementary material available at 10.1186/s44350-025-00004-7.

Keywords: Animal model; Granuloma; Mycobacterium avium complex; Plethysmography; Respiratory infection.

Fig. 1

Time course infection of C3HeB/FeJ mice infected with MAC2285R. Lung (A), spleen (B) and liver (C) bacterial burden (y-axis, log₁₀ CFU) in C3HeB/FeJ mice infected by intrapulmonary aerosol with high (10⁵ CFU) and low (10³ CFU) dose of MAC2285R was measured 4-weekly for 12 weeks. Mice infected with low-dose inoculum exhibited a rise in lung CFU between weeks 4–8 (p < 0.05) with modest extrapulmonary dissemination. Bacterial burden then reduced in lung, spleen and liver between weeks 8–12. Mice infected with high-dose inoculum exhibited largely stable pulmonary bacterial burden over 12 weeks, with liver but not splenic dissemination. Histological lesion scoring of lungs was performed at weeks 4, 8 and 12 (D), calculated as the proportion of infected area over the total lung area per animal (n = 3–4 per group). Inflammatory changes were mild, as demonstrated in representative histological heat maps at week 12 post-infection (E + F). Higher magnification revealed small aggregates of macrophages and epithelioid cells (G, H&E stained) with scattered intracellular bacteria (H, red arrows, Ziehl-Neeson staining). Data is representative of two studies (n = 3–8 per time point) and displayed as mean ± SD (A-C) or as individual data points with median line (D). Differences between time points were analysed by unpaired t-test (A-C) or by one-way ANOVA with Turkey’s multiple comparison test (D)

Fig. 2

Whole body plethysmography in C3HeB/FeJ mice infected with MAC2285R. Mice infected with high-dose inoculum MAC2285R exhibited evidence of increasing respiratory effort over 12 weeks’, evidence by significantly higher respiratory rate (A), tidal volume (B), inspiratory flow rate (C) and expiratory flow rate (D) (p < 0.0001 for all). Mice with low dose infection did not exhibit any significant differences in these parameters compared to uninfected mice. MAC infection led to reduced inspiration time (E) and end-inspiratory pause (F), regardless of infectious dose, but only high dose infection was associated with reduced expiration time (G) and end-expiratory pause (H) (p < 0.0001). Data points displayed as 3-point moving mean for time-course graphs (with error bars removed for clarity). Data displayed mean + SD for AUC graphs and analysed by one-way ANOVA with Turkey’s multiple comparison test. Data representative of one study with n = 4 per group. **** p < 0.0001; ns, not significant; Un, uninfected; LDI, low dose inoculum; HDI, high dose inoculum

Fig. 3

Time course infection of C3HeB/FeJ mice infected with MAC104. Lung (A), spleen (B) and liver (C) bacterial burden (y-axis, log₁₀ CFU) in C3HeB/FeJ mice infected by intrapulmonary aerosol with high (10⁵ CFU) and low (10³ CFU) dose of MAC101 was measured 4-weekly for 12 weeks (with the exception of week 4 in the low-dose inoculum group for which there were insufficient mice for analysis). Low-dose inoculum led to a proliferative lung infection over 8 weeks (p < 0.01), followed by plateau. There was no detectable splenic or hepatic dissemination. High dose inoculum generated a stable pulmonary infection and extrapulmonary dissemination for the first 8 weeks, with a modest reduction in lung bacterial burden between weeks 8–12 (p = 0.05). Histological lesion scoring of lungs was performed at weeks 4, 8 and 12 (D), calculated as the proportion of infected area over the total lung area per animal (n = 3–4 per group). Inflammatory changes were mild, as demonstrated in representative histological heat maps at week 12 post-infection (E + F). Higher magnification revealed small aggregates of macrophages and epithelioid cells (G, H&E staining) with occasional clusters of intracellular bacteria (H, red arrow, Ziehl-Neeson staining). Data is representative of two studies (n = 3–8 per time point) and displayed as mean ± SD (A-C) or as individual data points with median line (D). Differences between time points were analysed by unpaired t-test (A-C) or by one-way ANOVA with Turkey’s multiple comparison test (D)

Fig. 4

Whole body plethysmography in C3HeB/FeJ mice infected with MAC104. Mice infected with high-dose inoculum MAC104 exhibited evidence of increasing respiratory effort over 12 weeks’, evidence by significantly higher respiratory rate (A), tidal volume (B), inspiratory flow rate (C) and expiratory flow rate (D) (p < 0.0001). These mice also had significantly reduced inspiration time (E), end-inspiratory pause (F), expiration time (G) and end-expiratory pause (H) (p < 0.0001). Mice with low dose infection had similar respiratory response, but less pronounced. Data points displayed as 3-point moving mean for time-course graphs (with error bars removed for clarity). Data displayed mean + SD for AUC graphs and analysed by one-way ANOVA with Turkey’s multiple comparison test. Data representative of one study with n = 4 per group. ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant; Un, uninfected; LDI, low dose inoculum; HDI, high dose inoculum

Fig. 5

Time course infection of C3HeB/FeJ mice infected with MAC101. Lung (A), spleen (B) and liver (C) bacterial burden (y-axis, log₁₀ CFU) in C3HeB/FeJ mice infected by intrapulmonary aerosol with high (10⁵ CFU) and low (10³ CFU) dose of MAC101 was measured 4-weekly for 12 weeks. Histological lesion scoring of lungs was performed at weeks 4, 8 and 12 (D), calculated as the proportion of infected area over the total lung area per animal (n = 3–4 per group). Inflammatory changes were mild, as demonstrated in representative histological heat maps at week 12 post-infection (E + F). Higher magnification revealed small aggregates of macrophages and epithelioid cells (G, H&E staining) with scattered intracellular bacteria (H, red arrows, Ziehl-Neeson staining). Data is representative of two studies (n = 3–8 per time point) and displayed as mean ± SD (A-C) or as individual data points with median line (D). Differences between time points were analysed by unpaired t-test (A-C) or by one-way ANOVA with Turkey’s multiple comparison test (D)

Fig. 6

Whole body plethysmography in C3HeB/FeJ mice infected with MAC101. Mice infected with high-dose inoculum MAC101 did not exhibit evidence of increased respiratory effort compared to uninfected mice, as measured by respiratory rate (A), tidal volume (B), inspiratory flow rate (C) or expiratory flow rate (D). Neither was there significant difference in inspiration time (E), end-inspiratory pause (F), expiration time (G) or end-expiratory pause (H). In contrast, mice with low dose infection exhibited a period of reduced respiratory frequency, tidal volume and expiratory flow rates between weeks 5–10 post-infection (p < 0.05), which then recovered to levels comparable with uninfected controls by week 12. Data points displayed as 3-point moving mean for time-course graphs (with error bars removed for clarity). Data displayed mean + SD for AUC graphs and analysed by one-way ANOVA with Turkey’s multiple comparison test. Data representative of one study with n = 4 per group. * p < 0.05; ** p < 0.01; **** p < 0.0001; ns, not significant; Un, uninfected; LDI, low dose inoculum; HDI, high dose inoculum