Gulf war illness-related chemicals increase CD11b/c+ monocyte infiltration into the liver and aggravate hepatic cholestasis in a rodent model

Abstract

Gulf War Illness (GWI) is a chronic multisymptom disorder affecting veterans of the 1990-91 Gulf war. GWI was linked with exposure to chemicals including the nerve gas prophylactic drug pyridostigmine-bromide (PB) and pesticides (DEET, permethrin). Veterans with GWI exhibit prolonged, low-level systemic inflammation, though whether this impacts the liver is unknown. While no evidence exists that GWI-related chemicals are hepatotoxic, the prolonged inflammation may alter the liver's response to insults such as cholestatic injury. We assessed the effects of GWI-related chemicals on macrophage infiltration and its subsequent influence on hepatic cholestasis. Sprague Dawley rats were treated daily with PB, DEET and permethrin followed by 15 minutes of restraint stress for 28 days. Ten weeks afterward, GWI rats or naïve age-matched controls underwent bile duct ligation (BDL) or sham surgeries. Exposure to GWI-related chemicals alone increased IL-6, and CD11b⁺F4/80^- macrophages in the liver, with no effect on biliary mass or hepatic fibrosis. However, pre-exposure to GWI-related chemicals enhanced biliary hyperplasia and fibrogenesis caused by BDL, compared to naïve rats undergoing the same surgery. These data suggest that GWI patients could be predisposed to developing worse liver pathology due to sustained low-level inflammation of the liver when compared to patients without GWI.

Figure 1

Timeline of treatments with Gulf War Illness (GWI)-related chemicals/stress and bile duct ligation (BDL), and serum markers of liver damage. (A) Diagram of treatments timeline. (B) Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin were assessed in serum of GWI or naïve (unexposed) rats which were subject to BDL or sham surgery. p < 0.05. * BDL vs Sham; #, GWI vs naïve. (C) Hematoxylin and eosin (H&E) staining of liver sections from naïve and GWI-chemical exposed rats that underwent sham or BDL surgeries. Scale bar, 100 μM.

Figure 2

Measurement of intrahepatic bile duct mass (IBDM), cholangiocyte proliferation and apoptosis in GWI versus naïve rats that underwent sham or BDL surgeries. (A) Images of immunohistochemistry (IHC) for cholangiocyte marker cytokeratin 19 (CK-19). (B) Plot of data from image analysis: percent pixel area per field stained for CK-19 was measured for all four animal groups. (C) Plot of RT-qPCR data for CK-19 in the liver of GWI with sham or BDL versus naïve animals with sham or BDL surgeries. (D) IHC images of proliferating cell nuclear antigen (PCNA) in livers of GWI-treated animals that had sham or BDL surgeries as compared to naïve animals subjected to sham or BDL. Quantification of PCNA IHC images are shown in (E). (F) RT-qPCR of PCNA marker in livers of GWI and naïve rats subjected to sham or BDL surgeries. (G) Images of apoptotic cells (stained in brown) versus normal cells counterstained with methyl-green, in liver sections of GWI and naïve animals which had sham or BDL surgeries. Quantification of apoptotic cells in all groups is shown in (H). p < 0.05. * BDL vs Sham; #, GWI vs naïve. Scale bar, 100 μM.

Figure 3

Proinflammatory cytokines in the livers of GWI versus naïve rats subjected to sham or BDL surgery. Expression of: (A) interleukin-1 (IL-1β); (B) interleukin-6 (IL-6); and (C) tumor necrosis factor–alpha (TNFα) was determined by RT-qPCR assays of RNA isolated from the livers of GWI and naïve animals subjected to sham or BDL surgeries.

Figure 4

Density and localization of CD11b/c⁺ and CD68⁺ macrophages in the liver of GWI and naïve rats that underwent sham or BDL surgeries. (A) Immunofluorescence (IF) of CD11b/c⁺ macrophages (red) and cholangiocytes (green) versus nuclei (stained in DAPI, shown in blue). (B) Plot of image analysis data, showing the percentage of pixel area per field measured for CD11b/c positive cells. (C) Same as in (A) except that in red are shown CD68⁺ cells. (D) Plot of data from image analysis, showing the percentage of pixels per field for CD68⁺ cells. Scale bar, 100 μM.

Figure 5

Characterization of specific subgroups of Cd11b/c⁺ macrophages in the livers of GWI and naïve rats that underwent sham or BDL surgeries. Double labeling IF was performed in order to test the colocalization of CD11b/c⁺ with F4/80⁺ cells (A) and of CD11b/c⁺ with CD68⁺ cells (B). In red are shown CD11b/c⁺ cells in both (A,B) panels; in green are F4/80⁺ macrophages (A) or CD68⁺ macrophages (B). Nuclei were stained with DAPI and shown in blue in all images. (C) Results of RT-qPCR assays of M1 macrophages (C) and M2 macrophages (D) are shown for the following markers: NOS2 and CXCL9 in (C); Arg1 and Mrc2 in (D). The RNA was isolated from CD11b/c-positive cells which were fluorescence immuno-labeled and cut off the liver sections by laser capture microdissection. Scale bar, 100 μM.

Figure 6

Activation and proliferation of hepatic stellate (HSC) cells in GWI and naïve rats that underwent sham or BDL surgery. (A) Plot of data from RT-qPCR assays of alpha-smooth muscle actin (αSMA) in the livers of GWI and Naïve animals subjected to sham or BDL procedure. (B) IF of αSMA (red), CK-19 (green); cell nuclei were counterstained with DAPI (blue). (C) Plot of data from image analysis of αSMA expression in percent pixel area per field. (D) RT-qPCR results for desmin in livers of GWI and naïve rats that underwent sham or BDL surgery. (E) IF of desmin (red) and cholangiocytes (green) in liver sections of GWI and naïve rats with sham or BDL surgeries. Cell nuclei were counterstained with DAPI and shown in blue. (F) Plot of data from IF image analysis showing changes in desmin protein in the livers of various treatment groups. Desmin amount was expressed as percentage of pixel area per field. p < 0.05. * BDL vs Sham; #, GWI vs naïve. (G) Confocal microscopy colocalization of αSMA and desmin in livers of GWI and naïve rats when subjected to sham or BDL surgeries. Scale bar, 100 μM.

Figure 7

Hepatic fibrosis in GWI-chemicals treated rats as compared to naïve controls that underwent sham or BDL surgeries. (A–D) Plots of RT-qPCR data for markers of hepatic fibrosis: collagen 1A1(Col1A1), fibronectin 1 (FN1), matrix metalloproteinase 9 (MMP9), tissue inhibitor of metalloproteinases 1(TIMP1). (E) Images of liver sections stained with Sirius Red, a dye which specifically stains collagen I and collagen III fibrillary structures. (F) Plot of data from image analysis of Sirius Red staining of liver sections. p < 0.05. * BDL vs Sham; #, GWI vs Naïve. Scale bar, 100 μM.