PLD3 and PLD4 are single-stranded acid exonucleases that regulate endosomal nucleic-acid sensing

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Abstract

The sensing of microbial genetic material by leukocytes often elicits beneficial pro-inflammatory cytokines, but dysregulated responses can cause severe pathogenesis. Genome-wide association studies have linked the gene encoding phospholipase D3 (PLD3) to Alzheimer’s disease and have linked PLD4 to rheumatoid arthritis and systemic sclerosis. PLD3 and PLD4 are endolysosomal proteins whose functions are obscure. Here, PLD4-deficient mice were found to have an inflammatory disease, marked by elevated levels of interferon-γ (IFN-γ) and splenomegaly. These phenotypes were traced to altered responsiveness of PLD4-deficient dendritic cells to ligands of the single-stranded DNA sensor TLR9. Macrophages from PLD3-deficient mice also had exaggerated TLR9 responses. Although PLD4 and PLD3 were presumed to be phospholipases, we found that they are 5′ exonucleases, probably identical to spleen phosphodiesterase, that break down TLR9 ligands. Mice deficient in both PLD3 and PLD4 developed lethal liver inflammation in early life, which indicates that both enzymes are needed to regulate inflammatory cytokine responses via the degradation of nucleic acids.

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Fig. 1: Splenomegaly, IFN-γ-driven upregulation of MHC class II expression in peritoneal macrophages and other TLR9-dependent anomalies in Pld4–/– mice.
Fig. 2: Effect of deficiency in Pld4 and Tlr9 on infiltration of the liver by CD68+ macrophages.
Fig. 3: Analysis of the nuclease activity of PLD4 and PLD3.
Fig. 4: Exaggerated responses of Pld3–/– thioglycolate-elicited macrophages to an A-type TLR9 agonist and analysis of RNA expression of Pld3 and Pld4 in selected cell types.
Fig. 5: Stimulatory ability and stability of 2216 and its fragments in the presence or absence of PLD3 or PLD4.
Fig. 6: Liver inflammation and elevated inflammatory cytokine production in Pld3–/–Pld4–/– mice.
Fig. 7: Transfer of an inflammatory disease by transplantation of Pld3–/–Pld4–/– bone marrow.

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Acknowledgements

We thank B. Beutler (University of Texas Southwestern Medical Center) for Unc93b3d/3d mice; Y. Doyon (Centre Hospitalier Universitaire de Québec) for plasmid eSpCas9(1.1)51; and S. Kupriyanov and G. Martin (The Scripps Research Institute Genetics Core) and S. Head and P. Natarajan (The Scripps Research Institute NGS Core) for technical assistance. This project was supported by the US National Institutes of Health (R21AI126011, R21AI101692 and R37AI059714).

Author information

A.L.G. participated in many of the studies, identifying anomalies in PLD4- and PLD3-deficient mice and their myeloid cell cytokine responses and in the preparation of PLD3 and PLD4 proteins; D.H. elucidated the enzymatic function of PLD3 and PLD4, carried out all studies in 293 cells, and contributed to other studies; C.H. generated the Pld4fl/fl and Pld4–/– mutant mice and characterized many features of their phenotypes; A.M. cloned Pld4 cDNA and characterized Pld4 expression in the B cell lineage; V.T. and B.R.L. participated in the analysis of experimental autoimmune encephalomyelitis in Pld4–/– mice; P.D.S., T.R.B. and T.C.T. provided technical support; K.O. provided histological analysis of liver pathology; H.S.C., F.K., P.K., A. Zeitjian, R.L.S., M.B. and S.R.S. were student interns who participated in generating constructs for CRISPR-Cas9 mutagenesis and site-directed mutagenesis of Pld3 and Pld4; A. Zarpellon provided assistance with blood analysis using the IDEX Procyte DX system; B.C. assisted C.H. in the search for potential phospholipase activity of PLD4; E.S.P., M.O.K. and H.H. provided advice and assistance with DC studies (Fig. 1l–n) and data in Fig. 4d; J.T. and J.C.d.l.T. provided advice and assistance with RNA virus studies; M.D. assisted with PLD3 reagents and antibodies; L.T. provided advice on and assistance with the expression of PLD4 protein; A.L.G. and D.N. co-directed these studies; and A.L.G., D.H. and D.N. wrote the paper.

Correspondence to David Nemazee.

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Supplementary Figure 1 Generation and analysis of conditional Pld4 knockout mouse mutation and Pld3 null mutants.

(a) Design of targeting construct in relation to the wild type Pld4 locus, including restriction enzyme sites and location of hybridization probe. FRT recombinase recognition sites flanking the neomycin resistance gene (neo) are indicated with brown triangles and loxP sites are blue triangles. The constructs were designed such that CRE recombinase deletes Pld4 exons 4-6 containing the first HKD domain, and introduces a frame-shift. (b,c) Southern blot analysis of targeted embryonic stem cells, identifying clone 28 as correctly targeted. Experiment was performed twice for each identified cell line. (d) Western blot analysis of mouse PLD4 protein expression. Spleen lysates were analyzed using a rabbit polyclonal anti-PLD4 antiserum. Experiment was performed once. (e) Flow cytometry analysis of PLD4 expression in spleen DCs. DCs (CD11c+TCRbCD19) were gated as shown in the upper panels into pDCs (SiglecH+CD45RA+), CD8a DCs (CD11bCD8a+) and CD8 DCs (CD8CD11b+) and evaluated for intracellular PLD4 using MAb16 (lower panels) comparing Pld4fl/fl (red) and Pld4–/– (blue). Experiment was performed twice. (f) Numbers of sorted DC subsets isolated from spleens from 10 mice of indicated genotype. Error bars indicated standard deviation between three sorts. (g) Elevated MHCII but not CD86 in peritoneal macrophages of Pld4–/– compared to Pld4+/– mice n= 6/group. Statistics were determined using a two-tailed Student’s T test assuming equal variance. (h) Proportions of splenic B cell subsets from indicated genotypes. Mice were 8-10 weeks old, 6 mice/group. Marginal zone subset statistics were determined using a two-tailed Student’s T test assuming equal variance. (i) Normalization of MZ B cell frequency in the absence of IFN-γ. n=7,9. Statistics were determined using a two-tailed Student’s T test assuming equal variance. (j-n) Generation and analysis of Pld3 mutants. (j) Exon 9 sequence changes in mutant alleles of Pld3 transmitted by different founder mice. Note some lines had two independent mutant alleles. (k) Immunoblot of PLD3 protein expression in lysates from thioglycolate-elicited macrophages harvested from C57BL/6, Pld4–/–, or Pld3 Crispr Founder mice. This experiment was performed once. (l-n) Effect of Pld3 or Pld4 genotype on spleen weight (l), splenic NK cell percentage (m), and MHCII expression on resident peritoneal macrophages (n). Pld4–/– compared to Pld4fl/fl mice n= 4/group, Pld3+/– compared to Pld3–/– n=5/group. Bars show means and standard deviation and each point represents an individual animal. Statistics were determined using a two-tailed Student’s T test assuming equal variance. This experiment was performed twice.

Supplementary Figure 2 Analysis of experimental autoimmune encephalomyelitis induced in the indicated mouse strains.

EAE was induced as described in Methods and the clinical severity score was tracked over the indicated days. Each figure represents an independent set of experiments. (a-c) represent a series of studies carried out by the laboratory of BL in a distinct animal room and time period compared to (d-f), which were carried out by AG and TB using distinct reagents and mouse rooms. In experiments (a-e) mice of the indicated genotypes were challenged with MOG on d0. In (f) lethally irradiated B6.CD45.1 mice were first reconstituted with Pld4–/– or Pld4fl/fl bone marrow 10 weeks prior to EAE induction. In all cases, mean and standard error of the mean are presented. In (e), P value reflects comparison between Ifng–/–Pld4–/– and B6.CD45.1 scores. Statistics were determined using a two-tailed paired Student’s T test assuming equal variance.

Supplementary Figure 3 Splenic sorted CD8+ DCs produce IL-12 in response to 2216PS and VACV70 independent of the STING pathway.

WT and Tmem173–/– sorted CD8+ DCs were plated in duplicates, stimulated with the indicated agents overnight and cytokine release measured. (a) IFN-λ production, measured by ELISA. (b) IL-12p70 production, measured by Luminex assay. (c) Comparison of IL-12p70 production (measured by ELISA) of CD8+ cDC sorted from mice deficient in IFN-γ alone or lacking both IFN-γ and PLD4. Duplicate cultures shown. Experiment performed twice. (d) Sorted splenic CD8CD11bhi DCs produce IL-12p70 and IL-6 to indicated ligands. DCs plated in triplicate. Experiment performed twice. In all graphs, mean and standard deviation are shown. (c-d) Statistics were determined using a non-paired two-tailed Student’s T test assuming equal variance.

Supplementary Figure 4 LC-MS/MS analysis of bovine spleen phosphodiesterase II.

(a) An example of a peptide identification, with the peptide call in the upper left, the predicted fragments shown on the upper right, corresponding to the indicated m/z peaks in the chart. (b) Overview of the peptide matches to bovine PLD3, shown in red. The underlined peptide corresponds to the peptide shown in (a). (c) Complete list of all proteins identified by at least five unique peptides by LC-MS/MS analysis. PLD3 shown in bold. This experiment was performed once.

Supplementary Figure 5 Comparative efficiency of digestion of PLD4 and PLD3 for phosphorothioate (PS) and phosphodiester (PO) linked ODNs.

The indicated concentrations of mouse PLD3 or PLD4 were used to digest the indicated ODNs (2.5 μM) in 50 mM MEF pH 5.0, 125 mM NaCl at 37oC for 2 hours, electrophoresed on 20% polyacrylamide/TBE/urea gels and stained with Sybr gold. CpG-ODNs were provided in excess, 2.5 μM. This experiment was performed four times with similar results.

Supplementary Figure 6 Analysis of responses of Pld4–/– DCs, Pld3–/– thioglycolate-elicited macrophages and PLD3–/– human cell lines to TLR9 agonists.

(a-c) Responses of Pld4–/– DCs and Pld3–/– thioglycolate-elicited macrophages to 2216 fragments when complexed with Lyovec. Triplicate cultures of pDCs, CD8+ DCs and thioglycolate-elicited macrophages of the indicated genotypes were stimulated as in Fig 5 b-e except that the ligands were precomplexed with Lyovec at a concentration of 1 μg/ml. (a) CD8+ DCs; (b) pDCs, (c) macrophages. Mean and standard deviation are shown. This is representative of five experiments (a and b) and three experiments (c). Statistics were determined using an unpaired two-tailed Student’s T test assuming equal variance. (d) TLR9 responses of PLD3-deficient human cells to phosphorothioate (PS) or phosphodiester form of CpG-B (2006) or CpG-A (2216). PLD3 was knocked out in HEK-BluehTLR9 cells using CRISPR/Cas9, then individual, sequence-verified clones were assessed for TLR9 responses to the indicated ODNs. NFκB reporter activation was then measured. ODNs were used at 1 μM. Cells were plated in duplicates. Experiment performed three times with similar results. Statistics were determined using a two-tailed Student’s T test assuming equal variance comparing parental HEK-BluehTLR9 cells with PLD3KO.10. Mean and standard deviation are shown. (e) PLD3-deficient HEK-BluehTLR9 cells as in (d) stimulated with 2216 PS subfragments. Experiment was performed three times with similar results. Statistics were determined using a two-tailed Student’s T test assuming equal variance comparing parental HEK-BluehTLR9 cells with PLD3KO.10. Mean and standard deviation are shown. (f) IFN-α or IL-12p70 responses of Pld3+/– or Pld3–/– FLT3L-cultured DCs to listed TLR9 stimuli. Cells were plated in triplicates. Experiment was performed once. Mean and standard deviation are shown.

Supplementary Figure 7 Analysis of Pld3–/–Pld4–/– animals and irradiated recipients of bone marrow from Pld3–/–Pld4–/– animals.

(a) Weanling expected and observed genotype results from F2 crosses of either Pld3–/–Pld4+/– (left) or Pld3+/–Pld4–/– (right) parental breeding schemes. A Pearson’s Chi Square test was performed. (b) A 16 day-old litter from Pld3–/–Pld4+/– x Pld3+/–Pld4–/– breeding with genotype of pups shown. (c) Macroscopic image of liver from Pld3–/–Pld4–/– animal depicted in (b). (d) MHCII and CD86 expression levels of peritoneal macrophages isolated from bone marrow chimeric mice receiving bone marrow from either Pld3–/–Pld4+/– (red), Pld3+/–Pld4–/– (blue), or Pld3–/–Pld4–/– (green) donors. MHCII and CD86 expression levels of peritoneal macrophages from a control C57BL/6 animal (orange) are shown for comparison. Experiment performed once. Blood monocyte levels (e, i), serum cytokine IFN-γ (g), IFN-α (h), IL-6 (k) and chemokine CXCL10 (f), MCP3 (j) levels were determined from bone marrow chimeric mice receiving Pld3–/–Pld4+/– (dark grey), Pld3+/–Pld4–/– (light grey), or Pld3–/–Pld4–/– (white) bone marrow 8 weeks earlier (Dotted line represents level of sensitivity of assay). (e-k) Mean and individual mouse values are shown. Statistics were determined using a two-tailed Student’s T test assuming equal variance. Experiment was performed once.

Supplementary Figure 8 Gating strategies used to identify cellular subsets.

(a) DC subsets, (b) resident peritoneal macrophages, (c) isotype control staining for MHCII expression from resident peritoneal macrophages from control or Pld4–/– mice as gated in b, (d) bone marrow reconstituted B cells or (e) bone marrow reconstituted T cells.

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