Mice deficient in NKLAM have attenuated inflammatory cytokine production in a Sendai virus pneumonia model

Abstract

Recent studies have begun to elucidate a role for E3 ubiquitin ligases as important mediators of the innate immune response. Our previous work defined a role for the ubiquitin ligase natural killer lytic-associated molecule (NKLAM/RNF19b) in mouse and human innate immunity. Here, we present novel data describing a role for NKLAM in regulating the immune response to Sendai virus (SeV), a murine model of paramyxoviral pneumonia. NKLAM expression was significantly upregulated by SeV infection. SeV-infected mice that are deficient in NKLAM demonstrated significantly less weight loss than wild type mice. In vivo, Sendai virus replication was attenuated in NKLAM-/- mice. Autophagic flux and the expression of autophagy markers LC3 and p62/SQSTM1 were also less in NKLAM-/- mice. Using flow cytometry, we observed less neutrophils and macrophages in the lungs of NKLAM-/- mice during SeV infection. Additionally, phosphorylation of STAT1 and NFκB p65 was lower in NKLAM-/- than wild type mice. The dysregulated phosphorylation profile of STAT1 and NFκB in NKLAM-/- mice correlated with decreased expression of numerous proinflammatory cytokines that are regulated by STAT1 and/or NFκB. The lack of NKLAM and the resulting attenuated immune response is favorable to NKLAM-/- mice receiving a low dose of SeV; however, at a high dose of virus, NKLAM-/- mice succumbed to the infection faster than wild type mice. In conclusion, our novel results indicate that NKLAM plays a role in regulating the production of pro-inflammatory cytokines during viral infection.

Fig 1. NKLAM expression is increased during SeV infection.

A) NKLAM protein expression was assessed by Western blot. Each lane represents a single mouse. B) NKLAM protein expression by qPCR using lung homogenate during SeV infection. C) NKLAM mRNA expression in isolated SeV-infected mouse tracheal epithelial cells was determined by qPCR. D) NKLAM expression in infected (D4 and D7) tracheal homogenate was determined by qPCR. E) WT BMDM were treated with 100 μg/ml poly(I:C) for times indicated and NKLAM expression was assessed by qPCR. The mRNA levels (mean ± SD) are expressed relative to PBS-treated mice or unstimulated BMDM. n = 3 mice per group; * p < 0.05. F) WT and NKLAM^-/- BMDM were treated with 100 μg/ml poly(I:C) for 18h and NKLAM expression was determined by Western blot. Data are representative of two independent experiments.

Fig 2. NKLAM^-/- mice experience less weight loss during SeV infection.

All mice were infected with SeV (500 pfu/gram body weight) and their weight was monitored daily for 14 days. PBS (30 μL) was administered to uninfected control mice. n = 10 mice per group; p = 0.0004, Two-factor ANOVA.

Fig 3. NKLAM^-/- mice have a significantly lower viral load during SeV infection.

Mice were infected with 500 pfu SeV/gram body weight and homogenized tracheal (A) or lung tissue (B) was used to determine SeV viral load by qPCR. * p < 0.05; n = 3–8 mice per group; comparing NKLAM^-/- and WT mice at each day post-infection. C) mTEC were infected with SeV at an MOI of 0.1 for 72h and the SeV genome expression was determined by qPCR. Data represents three replicates.

Fig 4. SeV-induced STAT1 protein expression and STAT1 and NFκB p65 phosphorylation are lower in NKLAM^-/- than in WT mice.

WT and NKLAM^-/- mice were infected with 500 pfu SeV/gram body weight. A) Equal amounts (20 μg) of lung homogenate protein were then probed for pSTAT1(701) and STAT1. B) Graphical depiction of pSTAT1/STAT1 densitometric ratio from A. Quantitative PCR was used to determine the relative expression of STAT1 in infected lungs (C). The mRNA levels (mean ±SD) are expressed relative to PBS-treated mice. * p < 0.05; n = 3 mice per group. D) Equal amounts of lung homogenate protein were probed for phospho-NFκB p65 (S536) and p65. E) Graphical depiction pp65/p65 densitometric ratio of D. Each lane of Western blot data represents a single mouse. * p < 0.05; comparing NKLAM^-/- and WT mice at each day post-infection. n = 3 mice per group. ns; non-specific band. F) Isolated lung fibroblasts were treated with 100 μg/ml poly(I:C) for 120 min. Whole cell lysates were immunoblotted for pp65 and p65. G) Graphical representation of pp65/p65 densitometric ratio from F. Data are representative of 3 independent experiments. * p ≤ 0.05.

Fig 5. Cytokine expression is less in NKLAM^-/- mouse lung homogenate and NKLAM^-/- BMDM.

A) RNA isolated from lung homogenate from SeV-infected lungs was used to determine cytokine expression by qPCR. B) Isolated mTEC were incubated with SeV MOI 0.1 for 72h and IFNβ and IL-6 expression was determined by qPCR. C) BMDM were treated with 100 μg/ml poly(I:C) for 6 or 12 hours and cytokine expression was determined by qPCR. The mRNA levels (mean ± SD) are expressed relative to PBS-treated mice (n = 3 mice per group) or untreated mTEC or BMDM; * p < 0.05. Statistical comparisons are made between WT and NKLAM^-/- mice.

Fig 6. Examination of leukocyte infiltration into the lungs during SeV infection.

WT and NKLAM^-/- mice were uninfected (Unt) or infected with 500 pfu SeV/gram body weight. A) Lungs were dissociated into single cell suspensions were isolated at day 3, 5, and 7 post-infection and stained for CD45, CD3, NK1.1, CD11b, Gr-1 and F4/80. CD45⁺ cells were gated and the number of CD11b⁺/Gr-1⁺, CD3^-/NK1.1⁺ or CD11b⁺/F4/80⁺ cells was determined; * p < 0.05; n = 3 mice per group, comparing NKLAM^-/- and WT mice. B) Lungs from post-infection (p.i.) day 7 and 14 were formalin-fixed, embedded in paraffin and sections were stained with H&E. n = 2 mice per condition. Original magnification x50.

Fig 7. Autophagy marker LC3 expression is less in NKLAM^-/- mice and BMDM.

WT and NKLAM^-/- mice were infected with 500 pfu SeV/gram body weight and the conversion of LC3I to LC3II was determined by Western blot. A representative blot is shown in A. The ratio of LC3II/LC3I is shown graphically in B. n = 3 mice per group; * p < 0.05; comparing NKLAM^-/- and WT mice. C) WT and NKLAM^-/- BMDM were treated with 5 μg/ml rapamycin for 18h and stained for LC3 (green). Nuclei were counterstained with DAPI (blue). The number of LC3 puncta/cell was quantified using ImageJ. At least 250 cells were counted for each condition (D). White bar is 15 μm; * p ≤ 0.05. Data represent 3 independent experiments. E) RNA isolated from lung homogenate from SeV-infected lungs was used to determine LC3 expression by qPCR. F) WT and NKLAM^-/- BMDM were treated with 5 μg/ml rapamycin for 18h. LC3 protein expression was determined by Western blot and normalized to total protein using TCE. WT untreated values were set to 1. Data represent 3 separate experiments. SeV-infected lung homogenate was used to determine p62 expression by Western blot (G) and by qPCR (H). mRNA levels (mean ± SD) are expressed relative to PBS-treated mice; n = 3 mice per group; * p ≤ 0.05; comparing NKLAM^-/- and WT mice.

Fig 8. High-dose SeV infection is associated with a faster mortality rate in NKLAM^-/- mice.

Wild type and NKLAM^-/- mice were infected with 5 x 10³ (A) or 5 x 10⁴ (B) pfu SeV/gram body weight and monitored for signs of distress for 14 days. * p < 0.05 at day 8; Log-rank test for significance; n = 10 mice per group.