Clinical Significance of M1/M2 Macrophages and Related Cytokines in Patients with Spinal Tuberculosis

Abstract

Background: Macrophages are important immune cells involved in Mycobacterium tuberculosis (M.tb) infection. To further investigate the degree of disease development in patients with spinal tuberculosis (TB), we conducted research on macrophage polarization.

Methods: Thirty-six patients with spinal TB and twenty-five healthy controls were enrolled in this study. The specific morphology of tuberculous granuloma in spinal tissue was observed by hematoxylin-eosin (H&E) staining. The presence and distribution of bacilli were observed by Ziehl-Neelsen (ZN) staining. Macrophage-specific molecule CD68 was detected by immunohistochemistry (IHC). M1 macrophages play a proinflammatory role, including the specific molecule nitric oxide synthase (iNOS) and the related cytokine tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). M2 macrophages exert anti-inflammatory effects, including the specific molecule CD163 and related cytokine interleukin-10 (IL-10). The above markers were all detected by quantitative real-time PCR (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and IHC.

Results: Typical tuberculous granuloma was observed in the HE staining of patients with spinal TB. ZN staining showed positive expression of Ag85B around the caseous necrosis tissue and Langerhans multinucleated giant cells. At the same time, IHC results indicated that CD68, iNOS, CD163, IL-10, TNF-α, and IFN-γ were expressed around the tuberculous granuloma, and their levels were obviously higher in close tissue than in the distant tissue. RT-PCR and ELISA results indicated that IL-10, TNF-α, and IFN-γ levels of TB patients were also higher than those of the healthy controls.

Conclusion: The report here highlights that two types of macrophage polarization (M1 and M2) are present in the tissues and peripheral blood of patients with spinal TB. Macrophages also play proinflammatory and anti-inflammatory roles. Macrophage polarization is involved in spinal TB infection.

Figure 1

ZN staining and IHC in the TB lesion tissue. (a) ZN staining in tuberculous granuloma (×1000 oil immersion). (b) Ag85B is close to the TB lesion tissue, and the positive area is brown (×400). (c) Ag85B distance to the TB lesion tissue and negative expression (×400). (d) Quantitative comparison of Ag85B-positive area, ^∗∗P < 0.01.

Figure 2

H&E staining close to the TB lesion tissue and the distance to the TB lesion tissue. (a) Close to the TB lesion tissue H&E staining, with arrows pointing to the typical Langerhans multinucleated giant cells (×200). (b) Distance to the TB lesion tissue H&E staining (×200).

Figure 3

The IHC of macrophage polarization characteristic molecules and cytokines. (a1) IL-10 is close to the TB lesion tissue, and the positive area is brown (×400). (a2) IL-10 distance to the TB lesion tissue and negative expression (×400). (b1) TNF-α is close to the TB lesion tissue, and the positive area is dark yellow (×400). (b2). TNF-α distance to the TB lesion tissue and negative expression (×400). (c1) IFN-γ is close to the TB lesion tissue, and the positive area is dark yellow (×400). (c2) IFN-γ distance to the TB lesion tissue and negative expression (×400). (d1) CD68 is close to the TB lesion tissue, and the positive area is brown (×400). (d2) CD68 distance to the TB lesion tissue and the positive area is light yellow (×400). (e1) iNOS is close to the TB lesion tissue, and the positive area is brown (×400). (e2) iNOS distance to the TB lesion tissue and the positive area is light yellow (×400). (f1) CD163 is close to the TB lesion tissue, and the positive area is dark yellow (×400). (f2) CD163 distance to the TB lesion tissue and the positive area is light yellow (×400).

Figure 4

The IHC positive rates of macrophage polarization characteristic molecules and cytokines. Quantitative comparison of CD68-positive area, ^∗∗P < 0.01. Quantitative comparison of iNOS-positive area, ^∗∗∗∗P < 0.0001. Quantitative comparison of CD163-positive area, ^∗∗∗∗P < 0.0001. Quantitative comparison of IL-10-positive area, ^∗∗∗P < 0.001. Quantitative comparison of TNF-α-positive area, ^∗∗∗∗P < 0.0001. Quantitative comparison of IFN-γ-positive area, ^∗P < 0.05.

Figure 5

IL-10, TNF-α, and IFN-γ mRNA expression levels and cytokine levels. (a) The mRNA expression of IL-10, TNF-α, and IFN-γ in the leukocyte of spinal TB patients and control subjects. Significant differences: ^∗P < 0.05 and ^∗∗∗∗P < 0.0001. (b) Serum levels of IL-10, TNF-α, and IFN-γ in 36 patients with spinal TB and 25 control subjects. ^∗∗∗P < 0.001 and ^∗∗∗∗P < 0.0001.