Effector T cells control lung inflammation during acute influenza virus infection by producing IL-10

Abstract

Activated antigen-specific T cells produce a variety of effector molecules for clearing infection but also contribute to inflammation and tissue injury. Here we report an anti-inflammatory property of antiviral CD8+ and CD4+ effector T cells (T(eff) cells) in the infected periphery during acute virus infection. We find that, during acute influenza infection, interleukin-10 (IL-10) is produced in the infected lungs in large amounts--exclusively by infiltrating virus-specific T(eff) cells, with CD8+ T(eff) cells contributing a larger fraction of the IL-10 produced. These T(eff) cells in the periphery simultaneously produce IL-10 and proinflammatory cytokines and express lineage markers characteristic of conventional T helper type 1 or T cytotoxic type 1 cells. Notably, blocking the action of the T(eff) cell-derived IL-10 results in enhanced pulmonary inflammation and lethal injury. Our results show that antiviral T(eff) cells exert regulatory functions--that is, they fine-tune the extent of lung inflammation and injury associated with influenza infection by producing an anti-inflammatory cytokine. We discuss the potential implications of these findings for infection with highly pathogenic influenza viruses.

Figure 1. Influenza-specific T cells preferentially produce IL-10 and IFN-γ in the influenza-infected BALB/c lungs

(a) Quantitative RT-RCR analysis of IL-10 and IFN-γ transcript levels of purified CD3⁺and CD3⁻ cells in the lungs of mice 8 days p.i. (b, c) Quantitative RT-RCR analysis of IL-4, IL-17, IL-10 and IFN-γ transcript levels of purified CD8⁺(b) or CD4⁺(c) T cells in the lungs of mice 8 days p.i. (d) At day 8 p.i., lung, mediastinal lymph node (MLN) and spleen cells were restimulated in vitro with influenza infected BMDC and intracellular cytokine staining (ICS) was performed to measure the production of IL-10 and IFN-γ in gated CD8⁺ and CD4⁺ T cells. Numbers indicate the percentages of cytokine-positive cells in the gates within the total population. (e, f) Cells from MLN and lungs of mice 8 days p.i. were restimulated with flu-infected BMDC and ICS was performed to measure the production of IL-10 and IFN-γ in gated CD8⁺ (e) and CD4⁺ (f) T cells. The ratio of IL-10⁺ vs. IFN-γ⁺ CD8⁺ (e) or CD4⁺ (f) T cells and the mean fluorescence intensity (MFI) of IL-10 staining in IL-10⁺ CD8⁺ (e) or IL-10⁺ CD4⁺ (f) T cells in MLN and lungs are depicted. P values were determined by unpaired two-tailed student t test.

Figure 2. IL-10 producing CD8⁺ and CD4⁺ T cells are Type 1 effectors

BALB/c mice were infected with influenza. CD8⁺ (a–c) or CD4⁺ (e, f) cells from lungs of uninfected mice or mice 7 days p.i. with influenza were restimulated with influenza infected BMDC and the expression of T-bet (a, e), Foxp-3 (b, f) and a panel of effector or activation molecules (c) in uninfected lung CD8⁺ or CD4⁺ T cells (control) or IL-10 positive CD8⁺ or CD4⁺ T cells (IL-10⁺), IFN-γ single positive CD8⁺ or CD4⁺ T cells (IFN-γ SP), IL-10 and IFN-γ double negative CD8⁺ or CD4⁺ T cells (DN) from infected lungs were measured by ICS. Gray line: isotype control Ab staining. Numbers are MFI (a, e) or percentages of Foxp-3-positive cells in the gates within the total population (b, f). (d, g) Uninfected mice (control) and mice 6 days p.i. were injected with BrdU. 2 h later, mice were sacrificed and lung cells were restimulated with influenza-infected BMDC. The percentages of BrdU⁺ cells in CD8⁺ (d) or CD4⁺ (g) cells of uninfected lung (control) and the IL-10⁺, IFN-γ single positive (IFN-γ SP) and IL-10⁻ IFN-γ⁻ (DN) T cells of 6 days p.i. lung are depicted.

Figure 3. Kinetics of IL-10 producing CD8⁺ and CD4⁺ Te in vivo.

BALB/c mice were infected with influenza. At the indicated days p.i, lung cells were restimulated with influenza-infected BMDC. The production of IL-10 and IFN-γ by CD8⁺ and CD4⁺ T cells was measured by ICS. (a, c) FACS plots of IL-10 and IFN-γ production by CD8⁺ (a) or CD4⁺ (c) T cells in the lung Numbers indicate the percentages of cytokine-positive cells in the gates within the CD8⁺ (a) or CD4⁺ (c) T cell population. (b, d) The kinetics of accumulation (absolute numbers) of IL-10⁺ or IFN-γ⁺ CD8⁺ T cells (b) or IL-10⁺ or IFN-γ⁺ CD4⁺ T cells (d) in the lungs following influenza infection.

Figure 4. Te are the major source of IL-10 in vivo during influenza infection

BALB/c mice were infected with influenza (a–e). (a) At the indicated days p.i., IL-10 and IFN-γ levels in the BALF were measured by ELISA. (b) WT and Rag1^−/− mice were infected with influenza. At day 6 p.i., IL-10 and IFN-γ levels in the BALF were measured by ELISA. Rag control (Rag1^−/− mice uninfected), Rag Flu (Rag1^−/− mice infected with influenza), WT Flu (WT mice infected with influenza). (c) At day 6 p.i., IL- 10 and IFN-γ levels in the BALF were measured by ELISA. Control (uninfected mice), PBS (mice injected with PBS), α-CD8 (mice injected with CD8-specific mAb), α-CD4 (mice injected with CD4-specific mAb), α-CD8/α-CD4 (mice injected with CD8-specific plus CD4-specific mAbs). (d) At day 6 p.i., lung and MLN cells from control or CD4⁺ T cell depleted mice were restimulated in vitro with influenza-infected BMDC and ICS was performed to measure the production of IL-10 and IFN-γ in gated CD8⁺ T cells. Numbers indicate the percentages of cytokine-positive cells in the gates within the total population. (e) At day 6 p.i., mice were injected with monensin and the in vivo ICS assay was performed to measure the production of IL-10 and IFN-γ by CD8⁺ and CD4⁺ T cells in the lung in vivo. The numbers of IL-10⁺ CD8⁺, IL-10⁺ CD4⁺, IFN-γ⁺ CD8⁺ and IFN-γ⁺ CD4⁺ T cells are depicted. P values were determined by unpaired two-tailed student t test. (f) IL-10/eGFP reporter mice were infected with influenza. At day 6 p.i., the numbers of IL-10/eGFP⁺ CD8⁺ and IL-10/eGFP⁺ CD4⁺ T cells in the lungs, MLN and spleens are depicted.

Figure 5. IL-10R blockade in vivo results in increased mortality and accelerated death during influenza infection

(a) BALB/c mice were infected with influenza and treated with PBS, Rat IgG1 control mAb (Rat IgG1) or IL-10R-specific mAb (α-IL-10R). The survival of the mice was monitored daily. P value was determined by the Log-Rank survival test. (b) BALB/c mice were infected with influenza and treated with Rat IgG1 control mAb (Rat IgG1) or IL-10R-specific mAb (α-IL-10R). At day 6 and 8 p.i., airway (BALF) virus titers were assessed. P value was determined by unpaired two-tailed Student t test.

Figure 6. IL-10R blockade leads to lethal pulmonary inflammation during influenza infection

(a) BALB/c mice were infected with influenza and treated with IL-10R-specific mAb (α-IL-10R) or Rat IgG1 control mAb (Rat IgG1). At day 8 p.i., cytokines in the BALF were determined by Multi-plex cytokine array analysis. Shown are the fold changes of cytokines in the BALF of IL-10R mAb treated mice vs. those of cytokines in the BALF of Rat IgG1 treated mice. (b, c) BALB/c mice were infected with influenza and treated with IL-10R-specific mAb (α-IL-10R) or Rat IgG1 control mAb (Rat IgG1). At day 4, 6 and 8 p.i., lung inflammatory monocytic cells (b) and neutrophils (c) were determined by flow cytometry. P value was determined by unpaired two-tailed Student t test. (d, e) BALB/c mice were infected with influenza and treated with IL-10R-specific mAb (α-IL-10R) or Rat IgG1 control mAb (Rat IgG1). At day 4, 6 and 8 p.i., IL-12 p40 (d) and IFN-γ (e) levels in the BALF were determined by ELISA. P value was determined by unpaired two-tailed Student t test. (f, g) BALB/c mice were infected with influenza and treated with IL-10R-specific mAb (α-IL-10R) or Rat IgG1 control mAb (Rat IgG1). At day 8 p.i., lung cells were restimulated with flu infected BMDC and the numbers of IFN-γ⁺ CD4⁺ (f) and IFN-γ⁺ CD8⁺ (g) T cells were quantified by ICS. P value was determined by unpaired two-tailed Student t test. (h) BALB/c mice were infected with influenza and treated with IL-10R-specific mAb plus vehicle control (α-IL-10R + vechicle) or IL-10R-specific mAb plus corticosterone (α-IL-10R + steroids). The survival of the mice was monitored daily. P value was determined by the Log-Rank survival test.