Loss or blockade of myeloid TLR2 improves survival and reduces neutrophils and lung inflammation in influenza-infected neonatal mice
Synopsis
Combining human cord blood monocytes with a neonatal mouse influenza model, this study found that neonatal monocytes sustain higher TLR2 expression after LTA or IAV stimulation, while TLR2 gene deletion, anti-TLR2 antibody blockade, or myeloid-specific TLR2 deletion improved survival of IAV-infected neonatal mice (for example, 61% versus 25% survival for TLR2−/− versus wild-type neonates, and 46% versus 7% with anti-TLR2 antibody), accompanied by reduced neutrophils at 6 days post-infection, lower histopathology scores in myeloid conditional knockouts, and no further rise in IL-6, TNF-α, MCP-1, CXCL1, and CXCL2 from 3 to 6 days post-infection, suggesting that myeloid TLR2 signaling exacerbates neonatal susceptibility to respiratory viral infection by amplifying pulmonary inflammation.
IL-6 production in the supernatant of human neonatal cord blood mononuclear cells (n=4) and healthy adult PBMCs (n=3) following stimulation with (A) lipoteichoic acid at 2 μg/mL and 10 μg/mL or (B) PR8-IAV (MOI = 1.5). 2 experiments performed, data shown as Mean ± SEM. Toll-like receptor 2 (TLR2) surface expression on human monocytes from neonatal cord blood PBMCs and healthy adult PBMCs, following stimulation with (C) 2 μg/mL of LTA or (D) PR8-IAV (MOI = 1.5); values are presented as delta mean fluorescence intensities (MFIs) from unstimulated controls (n=5-13, 2-4 experiments performed, p<0.05*)
PubMedInterpretation
Human term neonatal monocytes show sustained and higher TLR2 expression after stimulation with the TLR2 ligand LTA and with IAV, while IL-6 production is similar to adults. Whereas neonatal TLR responses were previously framed as broadly impaired, this shifts the age difference toward distinct expression dynamics and provides a human-sample clue that TLR2 may be pathogenic in neonates. Cord blood mononuclear cells and adult peripheral blood mononuclear cells were compared for IL-6 by Luminex and TLR2 expression by flow cytometry after 2 and 10 μg/mL LTA and PR8-IAV; neonatal monocytes sustained higher TLR2 after LTA and showed a trend of higher TLR2 at 1 and 4 hours after IAV, with no significant changes in NK, B, or T cells.
Loss or blockade of TLR2 improves survival of IAV-infected neonatal mice, and this effect is age-specific. It repositions TLR2 from a protective pre-stimulation receptor to a receptor that drives post-infection pathology in neonates, while TLR2−/− adults showed weight loss similar to wild type. Three-day-old and 8-week-old TLR2−/− and C57BL/6 mice were infected intranasally with H1N1 PR8; TLR2−/− neonates survived at 61% versus 25% for wild type, and anti-TLR2 blocking antibody improved survival from 7% to 46% (p<0.01); TLR2−/− neonates had higher viral load at 1 day post-infection but similar loads at 3 and 6 days.
Neutrophils are pathogenic during neonatal IAV infection, and myeloid TLR2 signaling amplifies pro-inflammatory cytokines and chemokines. It links the survival benefit of whole-body TLR2 loss to neutrophil depletion and to the inflammatory phenotype of myeloid conditional knockout, indicating that myeloid TLR2 acts mainly by amplifying inflammation rather than by changing immune cell recruitment. Anti-Ly6G neutrophil depletion raised survival to 55% versus 25% for isotype controls, whereas anti-Gr-1 depletion of both monocytes and neutrophils did not improve survival (18% versus 16%); LysmΔtlr2 neonates had significantly lower pathology severity scores with roughly 1-20% of lung affected versus 10-30% in controls, and significantly lower IL-6, TNF-α, MCP-1, CXCL1, and CXCL2 at 6 days post-infection, with no differences in neutrophil or inflammatory monocyte recruitment.
Myeloid-specific MyD88 deletion also improves neonatal survival, and the survival difference from myeloid TLR2 deletion is not statistically significant, suggesting TLR2 is the predominant TLR driving pathology. In contrast to the prior observation that complete MyD88 knockout confers no survival benefit, this indicates that the pathogenic TLR signal is localized to myeloid cells. LysmΔmyd88 and LysmΔtlr2 were generated with lysozyme M promoter cre; PCR confirmed efficient deletion in alveolar macrophages, partial deletion in monocytes and neutrophils, and no deletion in epithelial cells or T cells; both conditional knockouts survived better than controls with viral loads comparable to controls.
Perspective
The results apply to 3-day-old neonatal mice infected intranasally with H1N1 PR8 and to in vitro stimulation of term (39-41 weeks gestation) cord blood monocytes; they are most useful for researchers and preclinical developers seeking to understand neonatal influenza immunopathology or to explore host-directed rather than direct antiviral strategies. Anti-TLR2 antibody was given 1 day before infection and continued every 48 hours, suggesting an intervention window covering the first week of infection; the neutrophil depletion and myeloid conditional knockout results point toward targeting the myeloid TLR2-neutrophil axis.
The authors note that only a subset of peripheral immune cells was examined for TLR2 expression and IAV response, and that lung-resident immune and non-immune cell responses remain to be studied; the compensatory pathways for viral control after TLR2 loss and the mechanism by which TLR2−/− neonates survive better despite higher early viral loads are not fully explained. Human data come from in vitro stimulation of cord blood monocytes, which differs from the in vivo infected lung environment; the timing, dose, and safety of anti-TLR2 blockade in infants have not been evaluated. In addition, some of this work was previously published in a doctoral thesis, so readers may note the extent of data overlap with that prior dissertation.