IL-4i1 Regulation of Immune Protection During Mycobacterium tuberculosis Infection

Abstract

Background: Interleukin 4 (IL-4i1)-induced gene 1 encodes L-phenylalanine oxidase that catabolizes phenylalanine into phenylpyruvate. IL-4i1 is mainly expressed by antigen-presenting cells (APCs), inhibits T-cell proliferation, regulates B-cell activation, modulates T cell responses, and drives macrophage polarization, but its role in bacterial infections is understudied.

Methods: We evaluated IL-4i1 deletion in macrophages and mice on infection with virulent H37Rv and W-Beijing lineage hypervirulent HN878 Mycobacterium tuberculosis (Mtb) strains. The bacterial growth and proinflammatory responses were measured in vitro and in vivo. Histopathological analysis, lung immune cell recruitment, and macrophage activation were assessed at the early and chronic stages of Mtb infection.

Results: IL-4i1-deficient (IL-4i1-/-) mice displayed increased protection against acute H37Rv, HN878 and chronic HN878 Mt infections, with reduced lung bacterial burdens and altered APC responses compared with wild-type mice. Moreover, "M1-like" interstitial macrophage numbers, and nitrite and Interferon-γ production were significantly increased in IL-4i1-/- mice compared with wild-type mice during acute Mtb HN878 infection.

Conclusions: Together, these data suggest that IL-4i1 regulates APC-mediated inflammatory responses during acute and chronic Mtb infection. Hence, IL-4i1 targeting has potential as an immunomodulatory target for host-directed therapy.

Keywords: Mycobacterium tuberculosis; IL-4i1; host-directed therapy; immunity.

Figure 1.

Interleukin 4i1 (IL-4i1) is highly expressed in HN878 Mycobacterium tuberculosis (Mtb)–infected interleukin 4 (IL-4)–stimulated macrophages and negatively regulates proinflammatory and bacterial killing effector functions. A, Bone marrow–derived macrophages (BMDMs) were generated from BALB/c mice and stimulated overnight with 100 U/mL of interferon (IFN) γ, IL-4, or IL-4/interleukin 13 (IL-13). Subsequently, cells were infected with HN878 Mtb strain at a multiplicity of infection of 1:5. The time-course expression of IL-4i1 (tags per million) transcripts after Mtb infection was determined using CAGE (Cap Analysis of Gene Expression) sequencing, and messenger RNA expression of IL-4i1 normalized to hypoxanthine guanine phosphoribosyl transferase (Hprt) was determined with quantitative polymerase chain reaction. B, Intracellular Mtb growth in wild-type (WT) and IL-4il–deficient (IL-4i1^−/−) BMDMs, measured 4 hours, 1 day, and 3 days after infection with H37Rv and HN878 Mtb strains. Abbreviation: CFUs, colony-forming units. C, Nitrite and cytokine production from Mtb-infected IL-4i1^−/− and WT BMDMs at 3 days after infection. Abbreviations: IL-1β, interleukin 1β; IL-12p40, interleukin 12p40; TNF, tumor necrosis factor. D, CFU burden and cell viability in IL-4–stimulated BMDMs treated with 2-, 10-, and 50-μmol/L benzoic acid (BzA) at the indicated time points; 0 days corresponds to 4 hours after infection. Data are representative of 2 independent experiments, and error bars denote means with standard errors of the mean (n=3–4). ∗P<.05; ∗∗P<.01; ∗∗∗P<.001 (2-tailed Student t test).

Figure 2.

Interleukin 4il (IL-4i1)–deficient (IL-4i1^−/−) mice displayed increased protection against acute H37Rv Mycobacterium tuberculosis (Mtb) infection with reduced pathology. A, Control littermate (wild-type [WT]) and IL-4i1^−/− mice were infected intranasally with 100 colony-forming units (CFUs) of H37Rv Mtb strain per mouse (n=5–8 per group). The mycobacterial burden in the lungs was measured by CFU count 21 days after infection. B, Lung weights were measured in H37Rv-infected mice 3 weeks after infection. C, Total cell numbers from single-cell suspensions of Mtb-infected lung tissues. D, Lung lesions were quantified by measuring the area of solid tissue versus ventilated air spaces from 3 deep-cut hematoxylin-eosin–stained lung sections per mouse (30 μm apart) and representative histopathological sections (original magnification ×20). Data in A–C are shown as the pool of 2 independent experiments, and data in D are representative of 2 independent experiments. ∗P<.05; ∗∗∗P<.001 (Student t test).

Figure 3.

Interleukin 4 il (IL-4i1) deletion in mice reduces interstitial macrophage recruitment in H37Rv Mycobacterium tuberculosis (Mtb)–infected lungs, increases nitric oxide production, but does not change T-cell numbers. A, Control littermate (wild-type [WT]) and IL-4i1–deficient mice (IL-4i1^−/−) were infected with 100 colony-forming units (CFUs) of H37Rv Mtb strain (n=5 per group). Percentage and total cell numbers of CD11c⁺SiglecF⁺ alveolar (Alv) and CD11b⁺F4/80⁺ interstitial (Int) macrophages, CD11b⁺CD11c⁺MHCII⁺ and CD11c⁺CD103⁺MHCII⁺ dendritic cells (DCs), and CD11b⁺Ly6G⁺ neutrophils 21 days after infection. B, Percentage and total cell numbers of CD19⁺ B cells, CD3⁺CD4⁺ T cells and CD3⁺CD8⁺ T cells in the lungs of infected mice. C, Percentage and total cell numbers of CD4⁺CD44⁻CD62L⁺ naive T cells, CD4⁺CD44⁺CD62L⁻ effector/effector memory T cells (T_eff), and CD4⁺CD44⁺CD62L⁺ central memory T cells (T_CM). D, interferon (IFN) γ, interleukin 1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor (TNF) α, interleukin 12p40 (IL-12p40), interleukin 12p70 (IL-12p70), and nitrite levels in lung homogenates. Dashed line indicates the assay’s limit of detection. Data are representatives of 2 independent experiments; error bars denote means with standard errors of the mean.∗P<.05; ∗∗P<.01 (Student t test).

Figure 4.

Interleukin 4i1 (IL-4i1)–deficient (IL-4i1^−/−) mice were resistant to HN878 Mycobacterium tuberculosis (Mtb) infection at 3 weeks. A, Control littermate (wild-type [WT]) and IL-4i1^−/− mice were infected intranasally with 100 colony-forming units (CFUs) of hypervirulent HN878 Mtb strain (n=3–5 per group). Mice were euthanized 21 days after infection to measure mycobacterial burden in the lungs. B, Spleen bacterial burdens in HN878-infected mice. C, Lung weights were measured in HN878-infected mice 3 weeks after infection. D, Tissue involvement was quantified from 2–3 deep-cut hematoxylin-eosin (HE)–stained lung sections per mouse (30 μm apart) and representative histopathological sections (original magnification ×20). Error bars denote means with standard errors of the mean. ∗P<.05 (Student t test).

Figure 5.

Interleukin 4i1 (IL-4i1) deficiency promotes the recruitment of “M1-like” restrictive macrophages and chemokine production in early HN878 Mycobacterium tuberculosis (Mtb) infection. A, Control littermate (wild-type [WT]) and IL-4i1–deficient (IL-4i1^−/−) mice were infected intranasally with 100 colony-forming unit (CFUs) of hypervirulent HN878 Mtb strain (n=5 per group). Mice were euthanized 12 days after infection. Mycobacterial burden in the lungs and lung weights were measured. B, Lung tissue involvement was quantified from 2–3 deep-cut hematoxylin-eosin–stained lung sections per mouse (30 μm apart) and representative histopathological sections (original magnification ×20). C, Percentage and total cell numbers of CD206⁺ and CD80⁺ alveolar (Alv; CD11c⁺SiglecF⁺) and interstitial (Int; CD11b⁺F4/80⁺) macrophages in infected WT and IL-4i1^−/− mice. D, IFN-γ, interleukin 1β (IL-1β), interleukin 6 (IL-6), transforming growth factor (TGF) β and nitrite levels in lung homogenates of infected WT and IL-4i1^−/− mice. The dashed line indicates the limit of detection of the assay; error bars denote means with standard errors of the mean. ∗P<.05; ∗∗P<.01 (Student t test).

Figure 6.

The decreased bacterial burdens in interleukin 4i1 (IL-4i1)–deficient (IL-4i1^−/−) mice are maintained in chronic stages of HN878 Mycobacterium tuberculosis (Mtb) infection. A, Control littermate (wild-type [WT]) and IL-4i1^−/− mice were infected intranasally with 100 colony-forming units (CFUs) of HN878 Mtb strain per mouse (n=6 per group). Mice were euthanized 8 weeks after infection. Mycobacterial burdens in the lungs and spleen were measured by CFU assay. B, Total lung weight (upper panel) and total cell numbers from single-cell suspensions (lower panel) were quantified. C, Lung tissue involvement was quantified from 4 deep-cut hematoxylin-eosin (HE)–stained lung sections per mouse (30–45 μm apart) and representative histopathological sections (original magnification ×20). D, Left, Percentages of alveolar (Alv) and interstitial (Int) macrophages, CD11b⁺ and CD103⁺ dendritic cells (DCs), and neutrophils in live cells 8 weeks after infection. Right, Percentages of B cells, CD4⁺ T cells, and CD8⁺ T cells in live cells. Detailed gating strategy can be seen in Supplementary Figure 5. Data shown are from a single experiment; error bars denote means with standard errors of the mean.∗P<.05; ∗∗P<.01; ∗∗∗P<.001 (Student t test).