Evaluation of Sex Differences in Murine Diabetic Ketoacidosis and Neutropenic Models of Invasive Mucormycosis

Abstract

There is increased concern that the quality, generalizability and reproducibility of biomedical research can be influenced by the sex of animals used. We studied the differences between male and female mice in response to invasive pulmonary mucormycosis including susceptibility to infection, host immune reaction and responses to antifungal therapy. We used diabetic ketoacidotic (DKA) or neutropenic mice infected with either Rhizopus delemar or Mucor circinelloides. The only difference detected was that when DKA mice were infected with M. circinelloides, female mice were more resistant to infection than male mice (median survival time of 5 vs. 2 days for female and male mice, respectively). However, a 100% lethality was detected among infected animals of both sexes. Treatment with either liposomal amphotericin B (L-AMB) or posaconazole (POSA) protected mice from infection and eliminated the difference seen between infected but untreated female and male mice. Treatment with L-AMB consistently outperformed POSA in prolonging survival and reducing tissue fungal burden of DKA and neutropenic mice infected with R. delemar or M. circinelloides, in both mouse sexes. While little difference was detected in cytokine levels among both sexes, mucormycosis infection in the DKA mouse model induced more inflammatory cytokines/chemokines involved in neutrophil (CXCL1) and macrophage (CXCL2) recruitment vs. uninfected mice. As expected, this inflammatory response was reduced in the neutropenic mouse model. Our studies show that there are few differences between female and male DKA or neutropenic mice infected with mucormycosis with no effect on the outcome of treatment or host immune response.

Keywords: Mucor; Rhizopus; mucormycosis; murine; sex.

Figure 1

Female and male DKA mice are equally susceptible to R. delemar infection. (A) Survival of female (F) or male (M) DKA mice combined from two independent experiments infected with R. delemar 99-880. Mice (n = 21 to 25 per group, except the uninfected control which had 7 for each sex) were made diabetic and infected. * p = 0.0003 for POSA-M vs. Placebo-M. ** p = 0.0008 for L-AMB-F vs. Placebo-F. *** p < 0.05 for L-AMB-M vs. Placebo-M or POSA-M. p values on the figure compare female and male mice within the same treatment. (B) Combined tissue fungal burden (qPCR) in lung and brain of mice infected with R. delemar. Mice (n = 20 per group from 2 experiments) were infected, treated and organs harvested at Day 4 post infection. Lung CFU: ^¥ p = 0.03 for Placebo-M vs. POSA-M, ^† p = 0.08 for Placebo-F vs. POSA-F, ^¥¥ p < 0.006 for L-AMB-M vs. Placebo-M or POSA-M, ^‡ p < 0.0001 for L-AMB-F vs. Placebo-F or POSA-F. Brain CFU: ^£ p = 0.135 for POSA-M vs. Placebo-M, ^Ω p = 0.02 for POSA-F vs. Placebo-F, ^Ψ p = 0.01 for L-AMB-M vs. Placebo-M or POSA-M, ^ΩΩ p < 0.0001 for L-AMB-F vs. Placebo-F and p = 0.1 for L-AMB-F vs. POSA-F.

Figure 2

Female DKA mice are slightly more resistant to M. circinelloides infection than male DKA mice. (A) Survival of female (F) or male (M) DKA mice combined from two independent experiments infected with M. circinelloides f. jenssenii DI15-131. Mice (n = 22–24 per group, except the uninfected control which had 10 for each sex) were made diabetic and infected. * p < 0.03 for POSA-F or POSA-M vs. Placebo of the corresponding sex. ** p < 0.02 for L-AMB-F or L-AMB-M vs. Placebo or POSA of the corresponding sex. p values on the figure compare female and male mice within the same treatment. (B) Combined tissue fungal burden (qPCR) in lung and brain of mice infected with M. circinelloides. Mice (n = 20 per group from 2 experiments) were infected, treated and organs harvested at Day 4 post infection. Lung CFU: ¥ p = 0.08 for POSA-M vs. Placebo-M, † p = 0.02 for POSA-F vs. Placebo-F, ¥¥ p < 0.0003 for L-AMB-M vs. Placebo-M or POSA-M, ‡ p < 0.001 for L-AMB-F vs. Placebo-F or POSA-F. Brain CFU: ^£ p = 0.1 for POSA-M vs. Placebo-M, Ω p = 0.02 for POSA-F vs. Placebo-F, Ψ p < 0.0001 for L-AMB-M vs. Placebo-M or POSA-M, ΩΩ p < 0.005 for L-AMB-F vs. Placebo-F or POSA-F.

Figure 3

Female and male neutropenic mice are equally susceptible to M. circinelloides infection. (A) Survival of female (F) or male (M) neutropenic mice combined from two independent experiments infected with M. circinelloides f. jenssenii DI15-131. Mice (n = 21 per group, except the uninfected control which had 10 for each sex) were made neutropenic and infected. * p = 0.002 for POSA F or POSA M vs. Placebo of the corresponding sex. ** p <0.001 for L-AMB-F or L-AMB-M vs. Placebo of the corresponding sex. p values on the figure compare female and male mice within the same treatment. (B) Combined tissue fungal burden (qPCR) in lung and brain of mice infected with M. circinelloides. Mice (n = 26 in each group from 2 experiments) were infected, treated and organs harvested at Day 4 post infection. Lung CFU: ^¥ p = 0.001 for POSA-M vs. Placebo-M, ^† p = 0.005 for POSA-F vs. Placebo-F, ^¥¥ p < 0.01 for L-AMB-M vs. Placebo-M or POSA-M, ^‡ p < 0.0001 for L-AMB-F vs. Placebo-F or POSA-F. Brain CFU: ^£ p = 0.75 for POSA-M vs. Placebo-M, ^Ω p = 0.53 for POSA-F vs. Placebo-F, ^Ψ p < 0.0001 for L-AMB-M vs. Placebo-M or POSA-M, ^ΩΩ p < 0.005 for L-AMB-F vs. Placebo-F or POSA-F.