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. 2020 Jan 21;10(1):777.
doi: 10.1038/s41598-019-57275-0.

Leaky-gut enhanced lupus progression in the Fc gamma receptor-IIb deficient and pristane-induced mouse models of lupus

Arthid Thim-Uam  1 , Saowapha Surawut  2 , Jiraphorn Issara-Amphorn  3 , Thiranut Jaroonwitchawan  3 , Pratsanee Hiengrach  3 , Piraya Chatthanathon  4 , Alisa Wilantho  5   6 , Naraporn Somboonna  4   5 , Tanapat Palaga  4 , Prapaporn Pisitkun  7 , Asada Leelahavanichkul  8   9
Affiliations

Leaky-gut enhanced lupus progression in the Fc gamma receptor-IIb deficient and pristane-induced mouse models of lupus

Arthid Thim-Uam et al. Sci Rep. .

Abstract

The influence of gut-leakage or gut-microbiota upon lupus progression was explored in 2 lupus mouse models. Pristane, administered in 4-wk-old wild-type (WT) female mice, induced lupus characteristics at 24-wk-old similar to the lupus-onset in FcGRIIb-/- mice. Gut-microbiota alteration was induced by co-housing together with the gavage of feces from 40-wk-old FcGRIIb-/- mice (symptomatic lupus). On the other hand, gut-leakage was induced by dextran sulfate solution (DSS) VSports手机版. DSS and gut-microbiota alteration induced high serum anti-dsDNA immunoglobulin (Ig) as early as 30 days post-DSS only in FcGRIIb-/- mice. DSS, but not gut-microbiota alteration, enhanced lupus characteristics (serum creatinine and proteinuria) in both lupus models (but not in WT) at 60 days post-DSS. Indeed, DSS induced the translocation of molecular components of gut-pathogens as determined by bacterial burdens in mesenteric lymph node (MLN), endotoxemia (gut-bacterial molecule) and serum (1→3)-β-D-glucan (BG) (gut-fungal molecule) as early as 15 days post-DSS together with enhanced MLN apoptosis in both WT and lupus mice. However, DSS induced spleen apoptosis in FcGRIIb-/- and WT mice at 30 and 60 days post-DSS, respectively, suggesting the higher impact of gut-leakage against spleen of lupus mice. In addition, macrophages preconditioning with LPS plus BG were susceptible to starvation-induced apoptosis, predominantly in FcGRIIb-/- cell, implying the influence of gut-leakage upon cell stress. In summary, gut-leakage induced gut-translocation of organismal-molecules then enhanced the susceptibility of stress-induced apoptosis, predominantly in lupus. Subsequently, the higher burdens of apoptosis in lupus mice increased anti-dsDNA Ig and worsen lupus severity through immune complex deposition. Hence, therapeutic strategies addressing gut-leakage in lupus are interesting. .

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Schematic diagrams of the experiment for survival analysis and gut permeability determination (A), exploration of the influence of dextran sulfate solution (DSS) induced gut-leakage in 8- and 24-wk-old mice (B) were demonstrated.
Figure 2
Figure 2
Survival analysis of FcGRIIb−/− and pristane mice (A), representative picture of visual characteristics of ascites in pristane mice (all 40-wk-old mice) (B) and representative lupus characteristics including serum creatinine (Cr), spot urine creatinine index (UPCI) and serum anti-dsDNA Ig in 8- and 24-wk-old mice in different groups (C–E) were demonstrated.
Figure 3
Figure 3
The survival analyses of mice with and without gut-leakage induced by dextran sulfate solution (DSS) in drinking water or control water of wild-type (WT), FcGRIIb−/− and pristane mice (A) (n = 10 per group) and time course of gut-leakage as measured by FITC-dextran assay of DSS mice (B) and mice in the protocol of co-housing (plus fecal gavage) with 40-wk-old WT (WT/ feces WT) or 40-wk-old FcGRIIb−/− (WT/feces FcGRIIb−/−) (C) (n = 6–9 per time-point) were demonstrated. In addition, time-course of serum anti-dsDNA Ig in mice with DSS or the co-housing (plus fecal gavage) in WT, FcGRIIb−/− and pristane mice (D–F) (n = 6–9 per time-point) were demonstrated.
Figure 4
Figure 4
The characteristics of wild-type (WT), FcGRIIb−/− and pristane mice with 60 days of dextran sulfate solution (DSS) (started at 8-wk-old; asymptomatic lupus) or water control drinking water as determined by renal injury (serum creatinine; Cr) (A), proteinuria (spot urine/creatinine) (B), systemic inflammation (serum IL-6) (C), and leaky-gut as determined by spontaneous serum (1→3)-β-D-glucan (BG) (D) and spontaneous endotoxemia (E) were demonstrated. In addition, these characteristics of mice with the protocol of co-housing (plus fecal gavage) by feces of 40-wk-old WT or 40-wk-old FcGRIIb−/− (F–J) and the comparison between DSS versus the co-housing (plus fecal gavage) (K–O) were indicated (n = 7–10 per group). *p < 0.05, #p < 0.05 vs. WT DSS.
Figure 5
Figure 5
Representative renal histopathology by Periodic Acid–Schiff (PAS) staining of wild-type (WT), FcGRIIb−/− and pristane mice at 16-wk-old with or without 60 days of dextran sulfate solution (DSS) in drinking water (A–F) and the injury score from glomeruli (G) and tubular injury (H) (see methods) were demonstrated (n = 4 per group). Glomerular hypertrophy were noted in FcGRIIb−/− and pristane with DSS.
Figure 6
Figure 6
Representative immunofluorescence pictures from glomeruli of wild-type (WT), FcGRIIb−/− and pristane mice at 16-wk-old with or without 60 days of dextran sulfate solution (DSS) in drinking water (A–F) were demonstrated.
Figure 7
Figure 7
Representative immunohistochemistry of apoptosis detection by active caspase-3 from spleen of wild-type (WT), FcGRIIb−/− and pristane mice at 16-wk-old with or without 60 days of dextran sulfate solution (DSS) in drinking water (A–F) were demonstrated.
Figure 8
Figure 8
Semi-quantitative analysis score of active caspase-3 in spleen of wild-type (WT), FcGRIIb−/− and pristane mice with or without 60 days of dextran sulfate solution (DSS) in drinking water (A) were demonstrated. In addition, the quantitative flow-cytometric analysis of splenocyte from these mice in term of necrotic cells (propidium iodide, PI +ve) (B), early apoptosis cells (Annexin V +ve, PI −ve) (C), late apoptosis cells (Annexin V +ve, PI +ve) (D), apoptotic macrophage in spleen (Annexin V +ve, F4/80 +ve) (E) and apoptotic B cell in spleen (Annexin V +ve, B220 +ve) were indicated (n = 4–6 per group).
Figure 9
Figure 9
Bacterial burdens in mesenteric lymph node (MLN) in wild-type (WT), FcGRIIb−/− and pristane mice with or without of dextran sulfate solution (DSS) in drinking water at different time-points (A), endotoxemia and serum (1→3)-β-D-glucan (BG) (B,C) were demonstrated (n = 5–6 per time-point for A–C). In addition, early apoptosis cells (Annexin V +ve, PI −ve), late apoptosis cells (Annexin V +ve, PI +ve), B cell apoptosis (Annexin V +ve, B220 +ve) and macrophage apoptosis (Annexin V +ve, F4/80 +ve) in MLN (D,E) were demonstrated (n = 5–6 per group for D,E).
Figure 10
Figure 10
The immune response of macrophage from wild-type (WT) and FcGRIIb−/− after stimulated in vitro with phosphate buffer solution control (PBS) or LPS with and without purified (1→3)-β-D-glucan (BG) after 24 h incubation was demonstrated (A). Quantitative flow-cytometric analysis of macrophage from WT and FcGRIIb−/− mice after LPS-stimulation with and without BG following by cell starvation (see methods) to determine necrotic cells (propidium iodide; PI +ve) (B), early apoptosis cells (Annexin V +ve, PI −ve) (C), late apoptosis cells (Annexin V +ve, PI +ve) (D) were demonstrated. In addition, other parameters of macrophage injury from these activations in TNF-related apoptosis-inducing ligand (TRAIL) (E), copy numbers of mitochondria DNA (mtDNA) (F), reactive oxygen species production as detected by dihydroethidium (DHE) (G) and total cell energy with ATP luminescence intensity (H) were indicated (independent triplicate experiments were performed). *p < 0.05; #p < 0.05 vs. same mouse strain in other experimental groups.
Figure 11
Figure 11
The characteristics of wild-type (WT), FcGRIIb−/− and pristane mice with 30 days of dextran sulfate solution (DSS) or water control drinking water (started at 24-wk-old; symptomatic lupus) as determined by serum anti-dsDNA Ig (A), renal injury (serum creatinine; Cr) (B), proteinuria (spot urine/creatinine) (C), systemic inflammation (serum IL-6) (D), and leaky-gut as determined by spontaneous serum (1→3)-β-D-glucan (BG) (E) and spontaneous endotoxemia (F) were demonstrated (n = 7–10 per group). *p < 0.05, #p < 0.05 vs. WT DSS.
Figure 12
Figure 12
Representative renal histopathology by Periodic Acid–Schiff (PAS) staining of 28-wk-old of symptomatic lupus (FcGRIIb−/− and pristane) and age-matched wild-type (WT) with or without 30 days of dextran sulfate solution (DSS) in drinking water (A–F) and the injury score from glomeruli (G) and tubular injury (H) (see methods) were demonstrated.
Figure 13
Figure 13
Representative immunofluorescence pictures from glomeruli of 28-wk-old mice of symptomatic lupus (FcGRIIb−/− and pristane) and age-matched WT (WT) with or without 30 days of dextran sulfate solution (DSS) in drinking water (A–F) were demonstrated.
Figure 14
Figure 14
Proposed hypothesis of gut-leakage enhanced lupus progression. Gut-leakage induces the translocation of gut bacteria, endotoxin (LPS) and (1→3)-β-D-glucan (BG) either in FcGRIIb−/− mice or wild-type (WT). However, inflammatory responses, apoptosis of immune cells and anti-dsDNA Ig production are more prominent in FcGRIIb−/− lupus mice due to the loss of inhibitory signaling.
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