Sex differences in the C57BL/6 model of Mycobacterium tuberculosis infection

Abstract

Globally, tuberculosis (Tb) notification data show a male-to-female ratio of 1.7 and higher, but the underlying reasons for the male bias remain elusive. Despite the well-known gender bias in human pulmonary Tb, a majority of experimental animal studies either do not separate and analyze data by sex or do not report the sex of their subjects at all. In the present study, we report increased male susceptibility in one of the most commonly used mouse models for Tb, C57BL/6 mice. Our study revealed that disease progression upon aerosol infection with Mycobacterium tuberculosis (Mtb) was accelerated in males resulting in increased morbidity and mortality compared to females. Elevated Mtb loads in males were associated with an early exaggerated pulmonary inflammatory response which likely was detrimental to the host, as reflected by exacerbated pathology and increased mortality. Our data emphasis the urgent need to include and separately analyze both sexes in future animal studies of Tb in order to appreciate the differences in immune responses and disease pathogenesis between males and females.

Figure 1

Decreased resistance of male C57BL/6 mice to Mtb infection. Male and female C57BL/6 were infected via aerosol with Mtb H37Rv and monitored for weight loss (A), development of clinical symptoms (B), and survival (C). Data pooled from two independent experiments are shown as mean ± SD (n = 8–10). Statistical analysis was performed by Mann-Whitney test (clinical score) or log rank test (survival).

Figure 2

Mycobacterial loads are increased in male lungs. Male and female C57BL/6 were infected via aerosol with Mtb H37Rv. At different time points, CFU were determined in organ homogenates. Data pooled from two independent experiments are shown as mean ± SD (n = 8–10). Statistical analysis was performed by Mann-Whitney test.

Figure 3

Histological characteristics of Mtb infected lungs. Male and female C57BL/6 were infected via aerosol with Mtb H37Rv and histologically evaluated after 152 days. (A) Granulomatous lesions in H&E stained lung sections (arrows: lymphoid aggregates). (B) Acid fast bacilli (arrows). (C) Immunohistochemical staining of iNOS. Representative micrographs from one mouse out of 10 mice per group are shown (original magnification × 40 in A and C; × 400 in B). (D) Quantitative assessment of total lung area affected by lesions (left graph) and the ratio of lymphoid aggregates to lesion area (right graph). (E) Quantitative evaluation of iNOS expression shown in (C. F) Immunohistochemical staining of neutrophils (Ly6B.2). Representative micrographs from one mouse out of 10 mice per group are shown (original magnification × 100).

Figure 4

Increased inflammatory responses upon Mtb infection in male lungs. Cytokine and chemokine levels were measured in lung lysates of female and male C57BL/6 mice on day 21 (A) and 152 (B) after Mtb H37Rv aerosol infection. Data pooled from two independent experiments are shown as mean ± SD (n = 9–10). Statistical analysis was performed by Mann-Whitney test. Correlations between cyto- or chemokines plotted versus bacterial burden in in lungs for day 21 (C) or d152 (D) in males and females. Each dot represents an individual sample (n = 9–10; females gray; males black).