Mycobacterium intracellulare induces a Th17 immune response via M1-like macrophage polarization in canine peripheral blood mononuclear cells

Abstract

Mycobacterium avium-intracellulare complex (MAC) is one of the most prevalent pathogenic nontuberculous mycobacteria that cause chronic pulmonary disease. The prevalence of MAC infection has been rising globally in a wide range of hosts, including companion animals. MAC infection has been reported in dogs; however, little is known about interaction between MAC and dogs, especially in immune response. In this study, we investigated the host immune response driven by M. intracellulare using the co-culture system of canine T helper cells and autologous monocyte-derived macrophages (MDMs). Transcriptomic analysis revealed that canine MDMs differentiated into M1-like macrophages after M. intracellulare infection and the macrophages secreted molecules that induced Th1/Th17 cell polarization. Furthermore, canine lymphocytes co-cultured with M. intracellulare-infected macrophages induced the adaptive Th17 responses after 5 days. Taken together, our results indicate that M. intracellulare elicits a Th17 response through macrophage activation in this system. Those findings might help the understanding of the canine immune response to MAC infection and diminishing the potential zoonotic risk in One Health aspect.

Figure 1

Heatmap of the canonical pathways related to macrophage activation in canine MDMs infected with M. intracellulare. The heatmap displays the canonical pathways related to (A) M1 macrophage and (B) M2 macrophage polarization. The color gradient reflects the predicted directions based on the z-score, where blue represents inhibition and red represents activation.

Figure 2

Gene expression analysis of canine MDMs infected with M. intracellulare. The expression level of genes related to (A) M1 and (B) M2 macrophages is presented as fold-change in mRNA expression. The mRNA expression was analyzed chronologically after canine MDMs were infected with M. intracellulare for 0–72 h. The mRNA expression in noninfected cells at 0 h was given a value of 1 as a reference for fold-change in expression. Each bar represents the mean ± SEM from three independent experiments in individual dogs. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 3

Ingenuity pathway analysis of the T helper cell response in M. intracellulare-infected canine MDMs. (A) The Th1 Pathway and (B) Th17 Activation Pathway were expressed at 72 h post infection. The individual nodes represent proteins with relationships represented by edges. The red and green colors indicate up- and downregulation based on the expression values, respectively. The activation states by z-scores are shown as follows: orange indicates activation, blue indicates inactivation, and uncolored nodes indicate genes that were not differentially expressed in this pathway.

Figure 4

Differential cytokine expression in mRNA level of monocyte-depleted PBMCs co-cultured with M. intracellulare-infected canine MDMs. (A) Flowchart of co-culture experiment for the T helper cell response. (B) The cytokines TNF-α, IFN-γ, and IL-12 (Th1); IL-4 and IL-13 (Th2); IL-6, IL-23, IL-1β, and IL-17 (Th17); and IL-10 and TGF-β (Treg) were quantified based on their fold changes of the mRNA expression in canine monocyte-depleted PBMCs co-cultured with M. intracellulare-infected MDMs. The mRNA expression in non-infected cells at 0 h was given a value of 1 as a reference for fold change in expression. Each bar represents the mean ± SEM from three independent experiments in individual dogs. *p < 0.05, **p < 0.01.

Figure 5

Cytokine expression in canine monocyte-depleted PBMCs co-cultured with canine MDMs after M. intracellulare infection. The concentration of IL-17A, IL-1β, IL-6, TNF-α, IL-12, IFN-γ, IL-10, and IL-4 was determined by ELISA. IL-4 and IL-12 were not detected. Cytokine expression in noninfected cells at each time point was used as a control. Each bar represents the mean ± SEM from three independent experiments in individual dogs. *p < 0.05, **p < 0.01.

Figure 6

IL-17- and IFN-γ-producing CD4⁺ T cells according to M. intracellulare infection and anti-CD3 stimulation. (A) Representative frequency of IL-17- or IFN-γ- producing canine CD4⁺ T cells in untreated lymphocytes (left), co-cultured with M. intracellulare-infected non-autologous MDMs (middle) or co-cultured with M. intracellulare-infected non-autologous MDMs with the anti-CD3 antibody (right). (B) Canine CD4⁺ T cells were co-cultured with M. intracellulare-infected non-autologous MDMs (#1, #2) and activated with anti-CD3 antibody (#1 + anti-CD3, #2 + anti-CD3). IL-17-expressing CD4⁺ T cells was analyzed by comparing untreated lymphocytes (filled). (C) The mean fluorescence intensity of IL-17 in co-cultured CD4⁺ T cells with M. intracellulare-infected non-autologous MDMs without (M. intracellulare) or with anti-CD3 antibodies (M. intracellulare plus anti-CD3), respectively. A dotted line indicates the expression of IL-17 in untreated lymphocytes. (D) In co-cultured with M. intracellulare-infected non-autologous MDMs with the anti-CD3 antibody, IL-17-expressing CD4⁺ T cells was analyzed by comparing isotype control (left). The co-cultured cells with non-infected MDMs with anti-CD3 antibody were used as a control (right).

Figure 7

IL-17- and IFN-γ-producing cells among canine CD4⁺ T cells in response to M. intracellulare. (A) Example of the flow cytometry gating strategy of IL-17- or IFN-γ- producing canine CD4⁺ T cells co-cultured with M. intracellulare-infected MDMs for 5 days (upper row) or noninfected MDMs (lower row). (B) Bar graph presenting the ratio of the proportion of IL-17-producing CD4⁺ T cells to IFN-γ-producing CD4⁺ T cells. Monocyte-depleted PBMCs were co-cultured with noninfected (control), M. intracellulare-infected, or LPS-stimulated MDMs. Each bar presents the mean ± SEM from five individuals.