Microbiota-dependent signals are required to sustain TLR-mediated immune responses
Lehn K. Weaver, Danielle Minichino, Chhanda Biswas, Niansheng Chu, Jung-Jin Lee, Kyle Bittinger, Sabrin Albeituni, Kim E. Nichols, Edward M. Behrens
Lehn K. Weaver, Danielle Minichino, Chhanda Biswas, Niansheng Chu, Jung-Jin Lee, Kyle Bittinger, Sabrin Albeituni, Kim E. Nichols, Edward M. Behrens
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Research Article Inflammation

Microbiota-dependent signals are required to sustain TLR-mediated immune responses

Abstract

Host-commensal interactions are critical for the generation of robust inflammatory responses, yet the mechanisms leading to this effect remain poorly understood. Using a murine model of cytokine storm, we identified that host microbiota are required to sustain systemic TLR-driven immune responses. Mice treated with broad-spectrum antibiotics or raised in germ-free conditions responded normally to an initial TLR signal but failed to sustain production of proinflammatory cytokines following administration of repeated TLR signals in vivo. Mechanistically, host microbiota primed JAK signaling in myeloid progenitors to promote TLR-enhanced myelopoiesis, which is required for the accumulation of TLR-responsive monocytes. In the absence of TLR-enhanced monocytopoiesis, antibiotic-treated mice lost their ability to respond to repeated TLR stimuli and were protected from cytokine storm–induced immunopathology. These data reveal priming of TLR-enhanced myelopoiesis as a microbiota-dependent mechanism that regulates systemic inflammatory responses and highlight a role for host commensals in the pathogenesis of cytokine storm syndromes.

Authors

Lehn K. Weaver, Danielle Minichino, Chhanda Biswas, Niansheng Chu, Jung-Jin Lee, Kyle Bittinger, Sabrin Albeituni, Kim E. Nichols, Edward M. Behrens

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Figure 5

Various antibiotic combinations result in protection from TLR9 cytokine storm, which correlates with low-stool DNA concentrations and distinct stool bacterial compositions.

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Various antibiotic combinations result in protection from TLR9 cytokine ...
A factorial study design was conducted to treat cohorts of mice with every combination of the 4-antibiotic cocktail found to protect mice from murine TLR9 cytokine storm (V, vancomycin; M, metronidazole; N, neomycin; A, ampicillin). C57BL/6 mice were pretreated with each antibiotic combination for 3 weeks prior to treatment of mice with 5 doses of the TLR9 agonist CpG1826 over the course of 10 days to induce TLR9 cytokine storm. Mice were sacrificed 24 hours after the fifth injection, and markers of disease were measured (see Supplemental Figures 3 and 4 for details). Stool was collected for 16S rRNA marker gene analysis prior to the first dose of CpG1826. The α-diversity of stool samples was measured using the Shannon index (A and E) and richness per 1000 reads (B and F). The DNA concentration of the stool samples was measured using the Quant-iT PicoGreen Assay Kit from Thermo Fisher Scientific (C and G). (D) Individual mice were categorized based on disease outcomes (splenomegaly, hepatomegaly, anemia, leukopenia, and thrombocytopenia) into groups of protected mice (4 of 5 disease parameters within 20% of PBS-treated mice or better), susceptible mice (4 of 5 disease parameters within 20%–30% of CpG-treated mice or worse), or partially protected mice (those mice with intermediate phenotypes). (E–H) Data display the indicated stool sample analysis by disease susceptibility grouping. (H) The β-diversity was assessed using unweighted UniFrac distances to compare the species composition of each sample principal coordinates analysis. The percentage in each axis represents the proportion of the total variance captured along the axis.