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Lactate production by Staphylococcus aureus biofilm inhibits HDAC11 to reprogramme the host immune response during persistent infection

Abstract

Staphylococcus aureus is a leading cause of biofilm-associated prosthetic joint infection (PJI), resulting in considerable disability and prolonged treatment. It is known that host leukocyte IL-10 production is required for S. aureus biofilm persistence in PJI. An S. aureus bursa aurealis Tn library consisting of 1,952 non-essential genes was screened for mutants that failed to induce IL-10 in myeloid-derived suppressor cells (MDSCs), which identified a critical role for bacterial lactic acid biosynthesis. We generated an S. aureus ddh/ldh1/ldh2 triple Tn mutant that cannot produce d- or l-lactate. Co-culture of MDSCs or macrophages with ddh/ldh1/ldh2 mutant biofilm produced substantially less IL-10 compared with wild-type S. aureus, which was also observed in a mouse model of PJI and led to reduced biofilm burden. Using MDSCs recovered from the mouse PJI model and in vitro leukocyte–biofilm co-cultures, we show that bacterial-derived lactate inhibits histone deacetylase 11, causing unchecked HDAC6 activity and increased histone 3 acetylation at the Il-10 promoter, resulting in enhanced Il-10 transcription in MDSCs and macrophages. Finally, we show that synovial fluid of patients with PJI contains elevated amounts of d-lactate and IL-10 compared with control subjects, and bacterial lactate increases IL-10 production by human monocyte-derived macrophages.

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Fig. 1: Lactate biosynthesis pathways induce MDSC and macrophage IL-10 production.
Fig. 2: Role for Ddh, Ldh1 and Ldh2 during S. aureus orthopaedic implant infection.
Fig. 3: S. aureus lactate promotes IL-10 production and regulates leukocyte infiltrates during PJI.
Fig. 4: S. aureus-derived lactate inhibits HDAC activity to regulate histone 3 acetylation and gene expression.
Fig. 5: S. aureus-derived lactate inhibits the negative regulator HDAC11 to augment leukocyte IL-10 production in an HDAC6-dependent manner.
Fig. 6: S. aureus-derived lactate induces IL-10 production by human monocyte-derived macrophages.

Data availability

The ChIP–seq and RNA-seq datasets are available in the GEO repository (accession number GSE135496). Source data are provided with this paper.

Code availability

All codes used are published programs, with citations for each provided in the references.

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Acknowledgements

This work was supported by the National Institutes of Health/National Institute of Allergy and Infectious Diseases grant no. P01AI083211 (Project 4 to T.K.) and grant no. R01AI125588 (to V.C.T.). The authors thank R. Fallet for managing the mouse colony. The UNMC DNA Sequencing Core receives partial support from the National Institute for General Medical Science (grant nos. INBRE–P20GM103427-14 and COBRE–1P30GM110768-01). Both the UNMC DNA Sequencing and Flow Cytometry Research Cores receive support from The Fred & Pamela Buffett Cancer Center Support Grant (grant no. P30CA036727).

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C.E.H. and T.K. conceived the study; C.E.H., M.E.B., K.J.Y., A.L.A. and T.K. designed experiments; and A.R.K. and D.K. provided expertise in the design, execution and data analysis for the ChIP–seq, ChIP–PCR and scRNA-seq experiments. S.S.C., A.A.A., C.M.G. and V.C.T. created the S. aureus lactate mutants used in the study. C.E.H., M.E.B., K.J.Y. and A.L.A. conducted the in vivo mouse PJI experiments. C.E.H. performed the in vitro biofilm–leukocyte co-culture experiments. E.S. and Y.L. provided the HDAC11 KO mice. C.E.H., D.K. and T.K. performed data analysis. T.K. procured funding for this work. C.E.H. and T.K. wrote the manuscript. All authors edited and approved the submission of this work.

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Correspondence to Tammy Kielian.

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Extended data

Extended Data Fig. 1 S. aureus lactate mutants do not display growth defects in liquid broth or biofilm in vitro.

a, S. aureus strains used in this study. b, The growth rate of S. aureus WT, Δddh, Δldh1/ldh2, and Δddh/ldh1/ldh2 was determined in brain-heart infusion broth over a 24 h period with constant agitation using a TECAN (7 biological replicates/strain). c, Strains were transduced with a sarA-GFP plasmid and grown for 4 days under static growth conditions in RPMI-1640 supplemented with 10% FBS, whereupon biofilm formation was visualized by confocal microscopy. Results are representative of two independent experiments, each with 4 biological replicates. Scale bars, 100 µm.

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Extended Data Fig. 2 Intracellular pH of MDSCs and macrophages is not dramatically altered by S. aureus-derived lactate during biofilm co-culture.

MDSCs or macrophages were labeled with BCECF-AM (10 µM) prior to co-culture with WT (n = 4 biological replicates/group) or Δddh/ldh1/ldh2 (n = 4 and 3 biological replicates for MDSCs and macrophages, respectively) biofilm for 2 h, whereupon intracellular pH was determined by flow cytometry based on a standard curve of known pH. Results shown are from one experiment.

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Extended Data Fig. 3 D- and L-lactate production during S. aureus orthopaedic infection.

(a) L- and (b) D-lactate were quantified in the implant-associated tissue of mice infected with WT, Δddh, Δldh1/ldh2, or Δddh/ldh1/ldh2 at days 3, 14, and 28 post-infection (mean ± SD; n = 5/group). The dashed line represents background in the assay as determined with tissues collected from animals receiving sterile implants at the same time points (n = 5 at days 3 and 28 and n = 4 at day 14). Results are representative of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; One-way ANOVA.

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Extended Data Fig. 4 The expression of select inflammatory mediators is independent of bacterial burden during S. aureus orthopaedic infection.

Cytokine levels were quantified in implant-associated tissue of mice infected with WT, Δddh/ldh1/ldh2, or a 1:1 ratio of WT and Δddh/ldh1/ldh2 at days 3, 14, and 28 post-infection. Results are combined from two independent experiments (mean ± SD; n = 8/group). *, p < 0.05; One-way ANOVA.

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Extended Data Fig. 5 IL-10 production during S. aureus orthopaedic infection is not influenced by host lactate.

Mice received daily i.p. injections of sodium oxamate (500 mg/kg/day) dissolved in 0.5% Hydroxypropyl Methylcellulose or vehicle (0.5% Hydroxypropyl Methylcellulose) beginning one day prior to infection with S. aureus WT (n = 10/group) or Δddh/ldh1/ldh2 (n = 10 or 9 for vehicle and oxamate, respectively). Mice were killed at day 14 post-infection to quantify (a) D-lactate, (b) L-lactate, and (c) IL-10 in implant-associated tissue. Results are combined from two independent experiments (mean ± SD). D- and L-lactate measurements are also reported at day 14 for mice that received sterile implants (n = 4/group). *, p < 0.05; **, p < 0.01; ***, p < 0.001; One-way ANOVA. NS, not significant.

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Extended Data Fig. 6 Sodium oxamate does not affect S. aureus growth or lactate production.

a, D- and (b) L-lactate were quantified in S. aureus WT and Δddh/ldh1/ldh2 biofilm in 96-well plates under static growth conditions in RPMI-1640 supplemented with 10% FBS over a 4 d period. The dotted lines represent daily medium changes. Results are from one experiment with 5 biological replicates. S. aureus was exposed to various concentrations of sodium oxamate during (c) growth in liquid broth (brain-heart infusion) beginning at time 0 (n = 6 biological replicates/group) or (d) throughout the 4-day biofilm maturation period (n = 5 biological replicates/group). Biofilm cultures were replenished daily with fresh medium (RPMI-1640 + 10% FBS) containing sodium oxamate. Results are presented as (c) OD600 or (d) Log10 colony forming units (CFU) per well. e, Quantification of D- and L-lactate from biofilm throughout the 4-day growth period, where the dotted lines represent daily medium changes (n = 4 biological replicates/group). All results are reported as mean ± SD.

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Extended Data Fig. 7 Effects of S. aureus lactate on orthopaedic implant biofilm infection are IL-10-dependent.

WT and IL-10 KO mice were infected with WT (n = 10) or Δddh/ldh1/ldh2 (n = 9) S. aureus, whereupon bacterial burden in (a) implant-associated tissue and (b) femur as well as (c) MDSC and (d) monocyte infiltrates were assessed at day 14 post-infection. Results represent the mean ± SEM of two independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; One-way ANOVA.

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Extended Data Fig. 8 S. aureus-derived lactate inhibits the negative regulator HDAC11 to augment leukocyte IL-10 production in a HDAC6-dependent manner.

MDSCs and macrophages from WT or HDAC11 KO mice were co-cultured with (a) WT or (b) Δddh/ldh1/ldh2 biofilm for 2 h ± HDAC6i (36 nM). IL-10 production was measured by cytometric bead array. Results represent the mean ± SEM of two independent experiments (n = 8 and 12 biological replicates for MDSCs and macrophages, respectively). **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; One-way ANOVA. Values for untreated leukocytes are the same as those presented in Fig. 5a,b because both HDAC6i and tubastatin A were tested at the same time.

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Extended Data Fig. 9 S. aureus-derived lactate preferentially inhibits HDAC11.

Purified active HDAC11 or HDAC6 were exposed to conditioned medium from WT or Δddh/ldh1/ldh2 biofilm for 30 min, whereupon HDAC activity was determined using a fluorescent HDAC substrate (deAc-FdL). Results are from one experiment with 4 biological replicates and are expressed as the percent change in HDAC activity compared to purified enzyme. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; One-way ANOVA. NS, not significant.

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Extended Data Fig. 10 Gating strategy to quantitate leukocyte populations in S. aureus implant-associated soft tissue.

Single cells were gated from the (a) total events using (b) FSC-A vs. FSC-H, followed by (c) exclusion of dead cells. (d) Live, CD45+ leukocytes were separated into (e) Ly6G+Ly6C+ vs. Ly6GLy6C+. (f) MDSC and neutrophil populations were identified based on CD11b expression, while (g) monocyte and macrophage populations were identified based on Ly6C and F4/80 expression, respectively.

Supplementary information

Supplementary Information

Supplementary Figs. 1–4 and Tables 1 and 2.

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Heim, C.E., Bosch, M.E., Yamada, K.J. et al. Lactate production by Staphylococcus aureus biofilm inhibits HDAC11 to reprogramme the host immune response during persistent infection. Nat Microbiol 5, 1271–1284 (2020). https://doi.org/10.1038/s41564-020-0756-3

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