Intratracheal Candida administration induced lung dysbiosis, activated neutrophils, and worsened lung hemorrhage in pristane-induced lupus mice

Abstract

Because the innate immunity might and fungi in the lungs might enhance the severity of lupus-induced diffuse alveolar hemorrhage (DAH), intraperitoneal pristane injection was performed in C57BL6 mice with intratracheal administration by Candida albicans or phosphate buffer solution (PBS). Despite the similar pristane-induced lupus (proteinuria, serum creatinine, and serum anti-dsDNA) at 5 weeks of the model, Candida administration worsened several characteristics, including mortality, body weight, serum cytokines (TNF-α and IL-6), and lung hemorrhage score, and cytokines in the lung tissue (TNF-α, IL-6, and IL-10), but not gut permeability (FITC-dextran assay), serum IL-10, immune cells in the spleens (flow cytometry analysis), and activities of peritoneal macrophages (polymerase-chain reaction). Although Candida administration reduced proteobacterial abundance and altered alpha and beta diversity compared with PBS control, lung microbiota was not different between Candida administration in pristane- and non-pristane-administered mice. Because of the prominent Gram-negative bacteria in lung microbiota and the role of neutrophils in DAH, lipopolysaccharide (LPS) with and without heat-killed Candida preparation was tested. Indeed, Candida preparation with LPS induced more severe pro-inflammatory neutrophils than LPS stimulation alone as indicated by the expression of several genes (TNF-α, IL-6, IL-1β, IL-10, Dectin-1, and NF-κB). In conclusion, the intratracheal Candida worsened pristane-induced lung hemorrhage partly through the enhanced neutrophil responses against bacteria and fungi. More studies on Candida colonization in sputum from patients with lupus-induced DAH are interesting.

Keywords: Fungi; Lung hemorrhage; Lupus; Microbiome; Pristane.

Fig. 1

Schema of the experiments (see method) (A) and characteristics of control, pristane injection, Candida administration (Candida), and pristane plus Candida (Pristane + Candida) as indicated by survival (B), bodyweight (C), lupus characteristics (urine protein creatinine index, serum creatinine, and serum anti-dsDNA) (D–F), gut permeability (FITC-dextran assay) (G), serum inflammatory cytokines (TNF-α, IL-6, and IL-10) (H–J) are demonstrated (n = 5–7/group).

Fig. 2

Characteristics of control, pristane injection, Candida administration (Candida), and pristane plus Candida (Pris + Can) as indicated by the representative lung histological pictures as stained by Hematoxylin and Eosin color (A), lung hemorrhage score (B), lung fungal abundance (C), and lung microbiome analysis, including alpha diversity (Chao-1 and Shannon index) with beta diversity (the Principle Coordinate Analysis (PCoA) based on Bray Curtis dissimilarity) (D–F) are demonstrated (n = 5–7/group for A–C and n = 3/group for microbiome analysis).

Fig. 3

Microbiome analysis from the lung of control, pristane injection, Candida administration (Candida), and pristane plus Candida (Pris + Can) as indicated by bacterial abundance in different levels (phylum, class, order, family, genus, and the average of genus) (A–F) with the selected graph presentation of only the high abundance groups in phylum (G) and genus (H) are demonstrated (n = 3/ group).

Fig. 4

Flow cytometry analysis of the spleens from control, pristane injection, Candida administration (Candida), and pristane plus Candida (Pris + Can) as indicated by the abundance of plasma cells (CD138 and B220 positive) (A), naïve Th cells (CD62L positive with CD44 negative) (B), total control memory T cells (CD3, CD44, and CD62L positive) (C), total Th cell (CD4 positive CD8 negative) (D), central memory Th cell (CD3, CD4, and CD62L positive) (E), effective memory Th cells (CD3 and CD4 positive with CD62L negative) (F), inducible T cell costimulatory (CD4 and ICOS positive) (G), central memory CD8 T cells (CD8 and CD62L positive) (H), adaptive B cells (GL7 and B220 positive) (I), IgM-producing B cell (IgM and B220 positive) (J), dendritic cells (CD80 and CD11c positive) (K), and activated dendritic cells (ICOS and CD11c positive) (L) are demonstrated (n = 5/ group). Lung tissue cytokines (TNF-a, IL-6, and IL-10) (M–O) are also demonstrated (n = 5/ group).

Fig. 5

Characteristics of tissue-resident macrophages (peritoneal macrophages) of mice from control, pristane injection, Candida administration (Candida), and pristane plus Candida (Pris + Can) as indicated by the biomarkers of M1 pro-inflammatory macrophages, including the expression of iNOS and IL-1β (using PCR) with CD80 and CD86 (flow cytometry), (A–D), M2 anti-inflammatory macrophages using Arg-1, Fizz-1, and TGF-β (PCR) with CD206 and CD163 (flow cytometry), (E–I), and expression of inflammatory genes (TNF-α, IL-6, IL-10, TLR-4, Dectin-1, and NF-κB) (J–O) are demonstrated (n = 5 /group).

Fig. 6

Characteristics of thioglycolate-induced neutrophils after the activation by lipopolysaccharide (LPS) or Candida preparation (see method) (Candida) or combined LPS plus Candida (LPS + Candida) or untreated control as indicated by expression of cytokine genes (TNF-α, IL-6, IL-1β, and IL-10) (A–D), TLR-4 gene (a LPS receptor) (E), Dectin-1 gene (a receptor of beta-glucan, an important component of Candida cell wall) (F), and NF-κB gene (an important transcriptional factor) (G) are demonstrated (results from isolated triplicated experiments).