Prevalence of hybrid TLR4+M2 monocytes/macrophages in peripheral blood and lung of systemic sclerosis patients with interstitial lung disease

Abstract

Introduction: Systemic sclerosis (SSc) is a complex autoimmune connective tissue disease characterized by microvascular damage, immune system reactivity and progressive fibrosis of skin and internal organs. Interstitial lung disease is the leading cause of death for SSc patients (SSc-ILD), and the process of lung fibrosis involves also circulating monocytes and alveolar macrophages.

Methods: Current study aimed to identify monocyte/macrophage phenotypes in lung and peripheral blood of SSc-ILD patients by immunostaining and flow cytometry, respectively. Single immunostaining was performed using primary antibodies against CD68 (pan-macrophage marker), CD80, CD86, TLR4 (M1 markers), CD163, CD204, and CD206 (M2 markers). Flow cytometry analysis included the evaluation of CD45, CD14, CD16 (monocyte lineage), CD1c (dendritic lineage), together with M1 and M2 activation markers on circulating monocytes. Protein synthesis of TLR4 and M2 markers was also investigated in cultured monocytes-derived macrophages (MDMs) from SSc-ILD patients by Western Blotting.

Results: Lung samples were obtained from 9 SSc-ILD patients (50 ± 9 years old) and 5 control non-SSc patients without lung fibrosis (58 ± 23 years old). Alveolar macrophages (CD68⁺ cells) showed a significantly higher positivity of M1 and M2 markers in SSc-ILD lung samples than in controls (p<0.05 for CD80, p<0.01 for CD86, p<0.001 for CD68, p<0.0001 for TLR4, CD163, CD204 and CD206). In CD68 positive areas of SSc-ILD samples, a significantly higher percentage of TLR4, CD163, CD204, and CD206 positive cells was observed compared to CD80 and CD86 positive cells (p<0.001 in both cases), suggesting the possible presence of hybrid TLR4⁺M2 macrophages (CD68⁺CD80^-CD86^-TLR4⁺CD163⁺CD204⁺CD206⁺cells) in SSc-ILD samples. A second cohort of 26 SSc-ILD patients (63 ± 14 years old) and 14 SSc patients without ILD (63 ± 19 years old) was recruited for flow cytometry analysis of circulating monocytes. Again, a significantly higher percentage of hybrid TLR4⁺M2 monocytes (CD1c^-CD80^-TLR4⁺CD163⁺CD204⁺CD206⁺cells) was found in SSc-ILD positive than SSc-ILD negative patients (p<0.05). Moreover, the protein synthesis of TLR4 and M2 markers was also found higher in cultured MDMs obtained from SSc-ILD patients than in MDMs from SSc patients without ILD and this increase was significantly higher for CD163 (p<0.05) and CD206 (p<0.01).

Conclusions: The presence of hybrid TLR4⁺M2 markers on both circulating monocytes and resident lung macrophages in SSc-ILD patients, is reported for the first time. Therefore, the detection of circulating hybrid TLR4⁺M2 monocytes in SSc-ILD might represent a further potential biomarker of progressive organ fibrosis, to be searched in blood samples of SSc patients.

Keywords: connective tissue diseases; fibrosis; interstitial lung disease; macrophages; monocytes; systemic sclerosis.

Figure 1

Gating strategy for the detection of circulating TLR4⁺M2 and M2 monocytes in systemic sclerosis patients (SSc pts) and healthy subjects (HSs) by flow cytometry. (A) Representative dot plot of leukocyte population using CD45 and physical parameters – Side scatter (SS) and Forward scatter (INT) – after removing cell debris (B) Flow cytometry scatter dot plot to identify circulating monocytes (CD45⁺CD14⁺CD16⁺cells) in the leukocyte population; (C) Flow cytometry scatter dot plot with quadrant regions of circulating CD163⁺CD206⁺ cells in the monocyte population; (D) CD204⁺CD163⁺CD206⁺cells in the monocyte population; (E) CD80^-TLR4^-CD204⁺CD163⁺CD206⁺cells (M2 monocytes) and CD80^-TLR4⁺CD204⁺CD163⁺CD206⁺cells (TLR4⁺M2 monocytes) in the CD204⁺CD163⁺CD206⁺ cells; (F, G) Flow cytometry scatter dot plot and related box plot representation of the percentage of TLR4⁺M2 monocytes (CD1c^-CD80^-TLR4⁺CD163⁺CD204⁺CD206⁺cells) and M2 monocytes (CD1c^-CD80^-TLR4^-CD163⁺CD204⁺CD206⁺cells) in HSs and SSc pts, excluding the presence of dendritic cells (CD1c⁺cells).

Figure 2

Masson’s trichrome staining of SSc-ILD lung samples (SSc-71, SSc-72, SSc-73, SSc-74, SSc-77, SSc-83, SSc-88, SSc-90, SSc-91) and control lung samples (CNT-A3, CNT-A5, CNT-A6, CNT-D8, CNT-D9). Blue color identifies collagen deposition, while violet color identifies cellular infiltrates in the stroma and in the alveoli (20x magnification).

Figure 3

Immunohistochemistry of a SSc-ILD sample (SSc-83) with 40x magnification. Representative Masson’s trichrome and immunostaining for CD68, M1 markers (CD80, CD86, TLR4) and M2 markers (CD163, CD204, CD206) are depicted. Red circles highlight areas rich of collagen in Masson’s trichrome staining and areas of co-localization of TLR4 positive cells and M2 markers (CD163, CD204, CD206).

Figure 4

Immunohistochemistry of a control lung sample (CNT-A6) with 40x magnification. Representative Masson’s trichrome and immunostaining for CD68, M1 markers (CD80, CD86, TLR4) and M2 markers (CD163, CD204, CD206) are depicted. M1 and M2 markers are virtually absent.

Figure 5

Percentage of positivity of all macrophage surface markers out of total cells in SSc-ILD lung samples versus controls (CNT). Three-quarters (almost 75%) of the cells found in the SSc-ILD lung samples belong to macrophage lineage. CD68, TLR4 (M1 marker), CD163 (M2 marker), CD204 (M2 marker) and CD206 (M2 marker) positive cells were found in the same areas. CD80 (M1 marker) and CD86 (M1 marker) positive cells were significantly less expressed than other markers investigated.

Figure 6

Flow cytometry analysis of circulating monocytes in systemic sclerosis (SSc) patients. Representative flow cytometry analysis to identify M2 monocytes (CD1c^-CD80^-TLR4^-CD163⁺CD204⁺CD206⁺cells) and TLR4⁺M2 monocytes (CD1c^-CD80^-TLR4⁺CD163⁺CD204⁺CD206⁺cells) in the peripheral blood of healthy subjects (HSs) and SSc patients with ILD (SSc-ILD) or without interstitial lung disease (SSc no-ILD). The related box plot representation of the percentage of these cells was included.

Figure 7

Evaluation by western blotting (WB) and related densitometric analysis of the protein synthesis of CD206, CD163, TLR4 and CD204 in cultures of monocyte-derived macrophages (MDMs) obtained from 5 voluntary healthy subjects (HS), 5 SSc patients without ILD (SSc no-ILD), and 11 SSc patients with ILD (SSc-ILD pts). The value of protein expression of CD206, CD163, TLR4 and CD204 was normalized to that of the corresponding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in cultured HS-MDMs, MDMs from SSc no-ILD, and MDMs from SSc-ILD. The resulting value of the protein expression of each molecule in cultured MDMs from SSc-ILD and from SSc no-ILD was compared with that obtained in cultured HS-MDMs (taken as unit value). The protein expression of each molecule obtained in cultured MDMs from SSc no-ILD and SSc-ILD represent the fold increase compared to the unit value of cultured HS-MDMs. Data are reported as median with a range of fold increase compared to HSs.