TLR3 absence confers increased survival with improved macrophage activity against pneumonia
Madathilparambil V. Suresh, … , Bethany B. Moore, Krishnan Raghavendran
Madathilparambil V. Suresh, … , Bethany B. Moore, Krishnan Raghavendran
Published December 5, 2019
Citation Information: JCI Insight. 2019;4(23):e131195. https://doi.org/10.1172/jci.insight.131195.
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Research Article Infectious disease

TLR3 absence confers increased survival with improved macrophage activity against pneumonia

Abstract

Toll-like receptor 3 (TLR3) is a pathogen recognition molecule associated with viral infection with double-stranded RNA (dsRNA) as its ligand. We evaluated the role of TLR3 in bacterial pneumonia using Klebsiella pneumoniae (KP). WT and TLR3–/– mice were subjected to a lethal model of KP. Alveolar macrophage polarization, bactericidal activity, and phagocytic capacity were compared. RNA-sequencing was performed on alveolar macrophages from the WT and TLR3–/– mice. Adoptive transfers of alveolar macrophages from TLR3–/– mice to WT mice with KP were evaluated for survival. Expression of TLR3 in postmortem human lung samples from patients who died from gram-negative pneumonia and pathological grading of pneumonitis was determined. Mortality was significantly lower in TLR3–/–, and survival improved in WT mice following antibody neutralization of TLR3 and with TLR3/dsRNA complex inhibitor. Alveolar macrophages from TLR3–/– mice demonstrated increased bactericidal and phagocytic capacity. RNA-sequencing showed an increased production of chemokines in TLR3–/– mice. Adoptive transfer of alveolar macrophages from the TLR3–/– mice restored the survival in WT mice. Human lung samples demonstrated a good correlation between the grade of pneumonitis and TLR3 expression. These data represent a paradigm shift in understanding the mechanistic role of TLR3 in bacterial pneumonia.

Authors

Madathilparambil V. Suresh, Vladislav A. Dolgachev, Boya Zhang, Sanjay Balijepalli, Samantha Swamy, Jashitha Mooliyil, Georgia Kralovich, Bivin Thomas, David Machado-Aranda, Monita Karmakar, Sanjeev Lalwani, Arulselvi Subramanian, Arun Anantharam, Bethany B. Moore, Krishnan Raghavendran

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Figure 1

Postmortem lungs from patients with KP show significant expression of TLR3.

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Postmortem lungs from patients with KP show significant expression of TL...
(A) Postmortem lung samples in patients with KP compared to normal lung tissue. Representative IHC images from a normal human lung stained with anti-TLR3 antibody and a lung with KP stained with anti-TLR3 antibody. Histopathological evaluation of postmortem lung samples (n = 11/samples) from controls and patients with KP (***P < 0.001 human normal lung vs. KP-infected lung). Interestingly, intense expression of TLR3 was also observed in 2 patients with deaths attributed to Acinetobacter infection (n = 10) (Table 1). (B) Immunocytochemistry: TLR3 expression in isolated alveolar macrophages from WT mice at 24 hours following KP. Samples were subjected to staining with TLR3 antibody (green) and nuclear staining with DAPI (blue) (n = 3/group). Statistical analysis was performed at each time point. Samples were analyzed using 2-tailed unpaired t test with Welch’s correction (*P < 0.05 WT uninjured vs. WT injured). (C) Capillary Western blot. TLR3 protein expression was determined at 24 hours following KP inoculation. Lung samples from 4 groups of mice (WT, TLR3–/– uninjured, WT + KP 24 hours, TLR3–/– + KP 24 hours were homogenized and subsequently lysed in RIPA buffer. Following Western immunoassay (Wes) protocol from Protein Simple, samples were loaded onto a plate and then tested using polyclonal TLR3 antibody (PA5-29619, eBioscience) and HRP-conjugated secondary antibodies (1:10, Anti-Rabbit Secondary Antibody, 042-206, Protein Simple). Data were analyzed using Protein Simple software to display bands. The box plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range.