Influenza virus-induced glucocorticoids compromise innate host defense against a secondary bacterial infection

Abstract

Multicellular organisms are continuously exposed to many different pathogens. Because different classes of pathogens require different types of immune responses, understanding how an ongoing immune response to one type of infection affects the host's ability to respond to another pathogen is essential for a complete understanding of host-pathogen interactions. Here, we used a mouse model of coinfection to gain insight into the effect of respiratory influenza virus infection on a subsequent systemic bacterial infection. We found that influenza infection triggered a generalized stress response leading to a sustained increase in serum glucocorticoid levels, resulting in a systemic suppression of immune responses. However, virus-induced glucocorticoid production was necessary to control the inflammatory response and prevent lethal immunopathology during coinfection. This study demonstrates that activation of the hypothalamic-pituitary-adrenal axis controls the balance between immune defense and immunopathology and is an important component of the host response to coinfection.

Figure 1

Bacterial burden is increased in influenza virus infected mice (A) Mice infected with influenza virus were infected with L. monocytogenes one day later, and four days after bacterial infection CFUs per gram of organ were determined for the spleen and the liver. (B) Mice were infected with 1000 PFUs of influenza virus 1 day, 3 days, or 5 days before infection with L. monocytogenes, and CFUs were determined four days after bacterial infection. (C) Mice were infected with 200 PFU of influenza virus 5 days before infection with L. monocytogenes and CFUs were determined 8 days after bacterial infection. Data are from at least 2 independent experiments with at least 4 mice in each group. (* p ≤ 0.05, ** p ≤ 0.001, *** p ≤ 0.0001, no * p >.05) see Figure S1

Figure 2

Bacterial burden is increased in co-infected IFNαR1^−/− and Rag2^−/− mice (A) Bacterial CFUs were measured in the liver and spleen 4 days after infection with L. monocytogenes in IFNαR1^−/− (B) or Rag2^−/− mice that were infected with 1000 PFU of influenza virus. (C) Viral titers were measured in the lungs of C57BL/6 mice infected with 100,000 PFUs and IFNαR1^−/− or Rag2^−/− mice that were infected with 1000 PFUs of influenza virus. Data are from at least 3 independent experiments with at least 3 mice in each group. (* p ≤ 0.05, ** p ≤ 0.001, no * p >.05) see Figure S3

Figure 3

Influenza virus infection causes immunosuppression (A) Splenocyte numbers were determined after infection with influenza and L. monocytogenes. (B) The levels of the cytokines IFN-γ and IL-6 in the serum were measured 1 and 4 days after infection with L. monocytogenes. Day 0 is prior to L. monocytogenes infection and one day after influenza infection (C) The areas of infiltrating cells per field were determined by blind scoring of H&E stained liver sections four days after infection with L. monocytogenes. (D) Gene expression in the liver was determined at 1 day, 2 days, and 4 days after bacterial infection in mice infected with influenza alone, with L. monocytogenes alone, or co-infected mice. In all experiments, mice were infected with 10,000 PFU of influenza one day before infection with L. monocytogenes. Data are from at least 3 independent experiments with at least 4 mice in each group. (* p ≤ 0.05, ** p ≤ 0.001, *** p ≤ 0.0001, no * p >.05) see Figure S2

Figure 4

Influenza virus-induced GC cause increased bacterial burden (A) Serum GC levels were measured in mice infected with influenza virus, L. monocytogenes, co-infected mice, and co-infected ADX mice. Day 0 indicates levels of serum GC levels prior to bacterial infection (B) Bacterial CFUs per gram were determined in the liver (top) and the spleen (bottom) 4 days after bacterial infection in sham-operated (SHAM) or adrenalectomized (ADX) mice infected with L. monocytogenes or mice co-infected with influenza and L. monocytogenes. In addition, CFUs were measured in mice infected with L. monocytogenes and treated daily with DEX. (C) Viral titres were measured in co-infected mice, co-infected SHAM mice, and co-infected ADX mice. In all experiments, mice were infected with 10,000 PFU of influenza one day before infection with L. monocytogenes. Data in (A) and (B) are from at least 3 independent experiments with at least 4 mice in each group, and data in (C) is from 2 independent experiments with 3-4 mice in each group. Bacterial CFUs comparing L. monocytogenes alone, co-infected, and co-infected ADX was repeated in 10 independent experiments with statistically significant results in each individual experiment, Mice with no detectable CFUs are indicated by symbols on the X-axis. (* p ≤ 0.05, ** p ≤ 0.001, *** p ≤ 0.0001, no * p >.05). see Figure S5

Figure 5

Removal of GC rescues virus-induced immunosuppression (A) Serum levels of IL-6 and IFN-γ and hepatic expression of (B) CCL7 and (C) Icam-1 were determined 1 day after infection with bacteria in mice infected with influenza virus, L. monocytogenes, co-infected mice, co-infected SHAM mice, co-infected ADX mice, and L. monocytogenes infected mice treated with DEX. (D) The amount of immune cell infiltrate into the liver, and (E) spleen weight were determined 4 days after bacterial infection in uninfected mice, mice infected with single pathogens, co-infected mice, co-infected SHAM mice, co-infected ADX mice, and L. monocytogenes infected mice treated with DEX. (F) The cytokines TNFα, IL-5, and IL-6 were measured in the serum 4 days after infection with L. monocytogenes, in mice that were infected with influenza and L. monocytogenes, co-infected ADX mice, co-infected SHAM mice, and singly infected controls. Data are from at least 3 independent experiments with at least 4 mice in each group. (* p ≤ 0.05, ** p ≤ 0.001, *** p ≤ 0.0001, no * p >.05) see Figure S6

Figure 6

ADX mice succumb to co-infection (A) Adrenalectomized mice infected with a sublethal dose of influenza virus (200 PFU) and five days later a sublethal dose of L. monocytogenes (5000 CFU) had decreased survival compared to singly infected ADX mice, singly infected mice, or co-infected mice. (B) The cytokines TNFα and IL-12p40 were elevated in the serum of co-infected ADX mice as early as one day after bacterial infection. Data are from at least 2 independent experiments with at least 4 mice in each group. (* p ≤ 0.05, *** p ≤ 0.0001, no * p >.05) see Figure S4

Figure 7

Lack of GC causes increased inflammation and immunopathology (A) Co-infected adrenalectomized mice have increased levels of IL-1β, IL-12p40, IL-6, IL-5, TNFα, and IFN-γ in the bronchoalveolar lavage fluid (BALF). (B) Co-infected ADX mice had increased lung pathology. (C) As a marker for epithelial cell damage albumin was measured in the BALF, and this was also increased in co-infected ADX mice. Data are from at least 2 independent experiments with at least 4 mice in each group. (* p ≤ 0.05, ** p ≤ 0.001, no * p >.05) see Figure S7