Germline gain-of-function myeloid differentiation primary response gene-88 (MYD88) mutation in a child with severe arthritis.

Myeloid differentiation primary response gene–88 (MYD88) encodes an essential adaptor protein connecting Toll-like receptor (TLR) and IL-1 receptor signaling to activation of IL-1 receptor–associated kinases (IRAKs). On receptor stimulation, MyD88 oligomerizes, causing recruitment and activation of IRAKs to form the myddosome (ie, MyD88 signaling complex),1Motshwene P.G. Moncrieffe M.C. Grossmann J.G. Kao C. Ayaluru M. Sandercock A.M. et al.An oligomeric signaling platform formed by the Toll-like receptor signal transducers MyD88 and IRAK-4.J Biol Chem. 2009; 284: 25404-25411Crossref PubMed Scopus (261) Google Scholar ultimately triggering activation of nuclear factor κB (NF-κB), interferon regulatory factor 7, or both.2Guven-Maiorov E. Keskin O. Gursoy A. VanWaes C. Chen Z. Tsai C.J. et al.The architecture of the TIR domain signalosome in the Toll-like receptor-4 signaling pathway.Sci Rep. 2015; 5: 13128Crossref PubMed Scopus (77) Google Scholar Germline loss-of-function mutations in MYD88 lead to immunodeficiency with recurrent pyogenic infections.3von Bernuth H. Picard C. Jin Z. Pankla R. Xiao H. Ku C.L. et al.Pyogenic bacterial infections in humans with MyD88 deficiency.Science. 2008; 321: 691-696Crossref PubMed Scopus (626) Google Scholar Somatic gain-of-function mutations contribute to certain B-cell malignancies.4Ngo V.N. Young R.M. Schmitz R. Jhavar S. Xiao W. Lim K.H. et al.Oncogenically active MYD88 mutations in human lymphoma.Nature. 2011; 470: 115-119Crossref PubMed Scopus (657) Google Scholar, 5Puente X.S. Pinyol M. Quesada V. Conde L. Ordóñez G.R. Villamor N. et al.Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia.Nature. 2011; 475: 101-105Crossref PubMed Scopus (1224) Google Scholar, 6Treon S.P. Xu L. Yang G. Zhou Y. Liu X. Cao Y. et al.MYD88 L265P somatic mutation in Waldenström's macroglobulinemia.N Engl J Med. 2012; 367: 826-833Crossref PubMed Scopus (923) Google Scholar Here we report a subject with a de novo germline gain-of-function MYD88 mutation who has severe destructive arthritis and an intermittent rash. The patient was a 14-year-old white girl with arthritis in small- to medium-sized joints since the age of 2 years, leading to severe bone destruction and periarticular growth arrest (Fig 1, A). Asymmetric arthritis often followed minor trauma, initially resembling septic arthritis, but synovial fluid culture results were negative, and antibiotics were ineffective. Two synovial biopsies of chronic arthritis revealed a marked neutrophilic infiltrate (not shown), which was unlike the lymphocytic pattern seen in patients with juvenile idiopathic arthritis. Magnetic resonance imaging revealed substantial synovial, periarticular, and intramedullary bone inflammation (Fig 1, B). The arthritis was unresponsive to nonsteroidal anti-inflammatory drugs, methotrexate, and corticosteroids, whereas biologics had been declined. Intermittent rashes began around the onset of her arthritis and were erythematous and maculopapular, generally lasting 1 to 2 weeks with spontaneous resolution (Fig 1, C). A recent skin biopsy from a dorsal hand lesion revealed interstitial granulomatous dermatitis. Whether this explains previous rashes is unclear. There was no history of unexplained fever, recurrent infection, or growth delay. The subject's C-reactive protein levels has been increased intermittently (<7 mg/L; normal, 0-4.99 mg/L), as has her osteocalcin levels (113.8-201.6 ng/mL; normal, 7.3-38.5 ng/mL), but with normal erythrocyte sedimentation rate, complete blood count, and serum immunoglobulin levels, including IgG subsets. Lymphocyte subsets (CD3, CD4/CD3, CD8/CD3, CD19, and natural killer cells) were normal, and no autoantibodies have been detected. For a summary of clinical findings, see Table E1 in this article's Online Repository at www.jacionline.org. The severe phenotype and inconsistencies with typical polyarticular juvenile idiopathic arthritis led us to pursue whole-exome sequencing, which revealed a heterozygous missense mutation in MYD88 (c.666T>G, p.Ser222Arg, or S222R) in the patient, but not other family members, which was also confirmed by using Sanger sequencing (see Fig E1, A, in this article's Online Repository at www.jacionline.org). The mutation was present in approximately 50% of reads from the patient's whole blood, CD14+ monocytes, cultured dermal fibroblasts, and patient-derived B-lymphoblastoid cells (EBV-LCLs), supporting a strong likelihood that it is a germline mutation (see Fig E1, B). Immunophenotyping revealed monocytes lacking CD16, which is known to shed during TLR activation,E1Picozza M. Battistini L. Borsellino G. Mononuclear phagocytes and marker modulation: when CD16 disappears, CD38 takes the stage.Blood. 2013; 122: 456-457Crossref PubMed Scopus (10) Google Scholar and identified a previously unreported CD123+CD11c+ dendritic cell population (Fig 1, D) that was negative for CD1c/blood dendritic cell antigen (BDCA)-1, CD303/BDCA-2, and CD141/BDCA-3 and the activated basophil marker CD203c (see Fig E2 in this article's Online Repository at www.jacionline.org). Additionally, CD19+CD20−CD27+CD38+ plasmablasts were absent, and CD20+CD19+IgD−CD27+ memory B-cell counts were decreased (Fig 1, D; see Table E1 for complete immunophenotyping data). There was a striking increase in Y705 signal transducer and activator of transcription 3 (STAT3) phosphorylation in unstimulated CD4+ and CD8+ T lymphocytes, a smaller increase in CD14+ monocytes, and a subpopulation of p-STAT3+CD19+ B lymphocytes, as has been reported in gain-of-function MYD88 mutation–positive malignancies.4Ngo V.N. Young R.M. Schmitz R. Jhavar S. Xiao W. Lim K.H. et al.Oncogenically active MYD88 mutations in human lymphoma.Nature. 2011; 470: 115-119Crossref PubMed Scopus (657) Google Scholar, 5Puente X.S. Pinyol M. Quesada V. Conde L. Ordóñez G.R. Villamor N. et al.Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia.Nature. 2011; 475: 101-105Crossref PubMed Scopus (1224) Google Scholar STAT3 phosphorylation was similar to that seen in control cells after IL-6 stimulation, except for a population of highly phosphorylated CD8+ T lymphocytes in the patient (Fig 1, E). Neutrophil surface CD11b, CD66b, and CD62L expression was similar to control values (data not shown), possibly because of chronic homeostatic changes in rates of surface antigen shedding to production or cell death/clearance. Peripheral monocyte gene expression revealed an interferon-regulated signature (NanoString data set available on request; Fig 1, F), and IL6 expression was also increased, although TNF expression was not (Fig 1, F). Whole-blood production of TNF-α and IL-6 from unstimulated cells from the patient was greater than control values (Fig 1, G), whereas these differences did not persist after TLR stimulation (data not shown). Amino acid residue 222 is located on the surface of the MyD88 Toll/IL-1 receptor (TIR) domain, which is required for oligomerization and myddosome formation.1Motshwene P.G. Moncrieffe M.C. Grossmann J.G. Kao C. Ayaluru M. Sandercock A.M. et al.An oligomeric signaling platform formed by the Toll-like receptor signal transducers MyD88 and IRAK-4.J Biol Chem. 2009; 284: 25404-25411Crossref PubMed Scopus (261) Google Scholar Oligomerization occurs through the BB-loop, α-helix E, and the C-terminus of the α-helix C.7Vyncke L. Bovijn C. Pauwels E. Van Acker T. Ruyssinck E. Burg E. et al.Reconstructing the TIR side of the myddosome: a paradigm for TIR-TIR interactions.Structure. 2016; 24: 437-447Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar Using molecular dynamics modeling, we found that the S222R mutation is predicted to introduce a novel R222-E245 salt bridge that, together with steric effects between the R222 and F248 side chains, induces a significant tilt of α-helix C (see Fig E3, A, in this article's Online Repository at www.jacionline.org). Similar to what is seen in the L265P-activating mutation,7Vyncke L. Bovijn C. Pauwels E. Van Acker T. Ruyssinck E. Burg E. et al.Reconstructing the TIR side of the myddosome: a paradigm for TIR-TIR interactions.Structure. 2016; 24: 437-447Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar this shift of the CD loop can promote MyD88 TIR-TIR symmetric interaction. Because the patient was heterozygous for S222R, we re-expressed equal amounts of wild-type (WT) and/or S222R MyD88 proteins with different epitope tags within the same MyD88-deficient (MyD88-KO) cell (see Fig E3, B). Unstimulated cells containing S222R MyD88 in conjunction with WT display 6- to 7-fold greater NF-κB activity compared with cells containing only WT or only S222R MyD88 (see Fig E3, C), which is consistent with previous findings.8Avbelj M. Wolz O.O. Fekonja O. Benčina M. Repič M. Mavri J. et al.Activation of lymphoma-associated MyD88 mutations via allostery-induced TIR-domain oligomerization.Blood. 2014; 124: 3896-3904Crossref PubMed Scopus (56) Google Scholar By using a proximity ligation assay, S222R exhibited a 1.8-fold enhanced interaction with WT (by using either tag combination) compared with interactions between 2 WT or 2 S222R-containing proteins (see Fig E3, D and E). These results demonstrate that the S222R mutation increases MyD88 interaction with WT MyD88, indicating that heterozygosity is sufficient for and might actually maximize MyD88 S222R gain-of-function effects. We investigated MyD88 pathway activation in dermal fibroblasts from patients, finding significantly increased gene expression of several neutrophil-attracting chemokines (NanoString data set available on request). Increases in CXCL1, CXCL5, and CXCL8 expression were confirmed by means of quantitative PCR (qPCR; Fig 2, A), and at the protein level, increases were confirmed for CXCL1 and IL-8 (see Fig E4, A, in this article's Online Repository at www.jacionline.org). The patient's fibroblast–conditioned media attracted healthy donor neutrophils to a greater extent than control-conditioned media (see Fig E4, B). MyD88 knockdown caused a significant reduction in CXCL8 expression (Fig 2, B), which was not seen with knockdown of the upstream TIR-containing adaptor protein (TIRAP; Fig 2, C). Downstream, the IRAK4 inhibitor AS2444697 caused a dose-dependent reduction in baseline CXCL8 expression, with little effect in control cells (Fig 2, D). Lastly, knockdown of the NF-κB p65 subunit significantly reduced CXCL8 to levels approaching values in control cells (Fig 2, E). Similar results were seen with CXCL1 and CXCL5 (data not shown). A20 (TNFAIP3), which negatively regulates the NF-κB pathway,E2Coornaert B. Carpentier I. Beyaert R. A20: central gatekeeper in inflammation and immunity.J Biol Chem. 2010; 284: 8217-8221Crossref Scopus (255) Google Scholar was upregulated in patient-derived EBV-LCLs (Fig 2, F) and fibroblasts (Fig 2, G). It has been reported previously that A20 upregulation in murine B lymphocytes containing MyD88 L265P limits NF-κB activation and proliferation.E3Wang J.Q. Jeelall Y.S. Beutler B. Horikawa K. Goodnow C.C. Consequences of the recurrent MYD88 (L265P) somatic mutation for B cell tolerance.J Exp Med. 2014; 211: 413-426Crossref PubMed Scopus (67) Google Scholar Knockdown of A20 values in the patient's fibroblast significantly increased CXCL8 expression (10.1-fold), as well as CXCL1 and CXCL5 (data not shown), and caused emergence of IL6 expression (Fig 2, H). These results suggest that A20 constrains, although incompletely, the increased activity of MyD88 S222R–regulated pathways producing proinflammatory mediators. The S222R mutation has been reported in a single DLBCL cell line (SUDHL2),4Ngo V.N. Young R.M. Schmitz R. Jhavar S. Xiao W. Lim K.H. et al.Oncogenically active MYD88 mutations in human lymphoma.Nature. 2011; 470: 115-119Crossref PubMed Scopus (657) Google Scholar in which it leads to overproduction of IL-6.E4Lam L.T. Wright G. Davis R.E. Lenz G. Farinha P. Dang L. et al.Cooperative signaling through the signal transducer and activator of transcription 3 and nuclear factor-kB pathways in subtypes of diffuse large B-cell lymphoma.Blood. 2008; 111: 3701-3713Crossref PubMed Scopus (285) Google Scholar We confirmed and extended the SUDHL2 result to include IL-8 (see Fig E5, A, in this article's Online Repository at www.jacionline.org) and demonstrate that patient-derived EBV-LCLs with MyD88 S222R also overexpress IL-6/IL-8 at the mRNA and protein level in an IRAK4-dependent manner (see Fig E5, B). SUDHL2, like other MYD88 mutations containing DLBCL cells, overexpress and hyperphosphorylate STAT3 (Fig E5, C), which is believed to be due to autocrine IL-6.E4Lam L.T. Wright G. Davis R.E. Lenz G. Farinha P. Dang L. et al.Cooperative signaling through the signal transducer and activator of transcription 3 and nuclear factor-kB pathways in subtypes of diffuse large B-cell lymphoma.Blood. 2008; 111: 3701-3713Crossref PubMed Scopus (285) Google Scholar Similarly, patient-derived EBV-LCLs exhibit increased p-STAT3 (Y705; see Fig E5, D), although STAT3 expression is not increased. To directly assess the role of autocrine IL-6, we first treated DLBCL U2932 cells (expressing WT MyD88) with SUDHL2-conditioned media, which caused increased p-STAT3 that could be partially blocked by the IL-6 receptor antagonist tocilizumab (TCZ; see Fig E5, E). Similarly, we exposed unrelated healthy control EBV-LCLs to EBV-LCL–condition media from the patient or her mother. Like SUDHL2 media, patient-conditioned media caused increased p-STAT3 levels in these cells, which could also be blocked partially with TCZ, demonstrating bioactive IL-6 secreted from the patient's B cells (see Fig E5, F). Both SUDHL2 and the patient's EBV-LCLs also overproduced IL-10 compared with control cells (data not shown), which could also be influencing Y705 p-STAT3 levels. This is the first report of a germline gain-of-function mutation in MYD88 (S222R) in a subject with destructive polyarthritis and rash. We show that S222R activates MyD88, confirming and extending previous observations,4Ngo V.N. Young R.M. Schmitz R. Jhavar S. Xiao W. Lim K.H. et al.Oncogenically active MYD88 mutations in human lymphoma.Nature. 2011; 470: 115-119Crossref PubMed Scopus (657) Google Scholar, 7Vyncke L. Bovijn C. Pauwels E. Van Acker T. Ruyssinck E. Burg E. et al.Reconstructing the TIR side of the myddosome: a paradigm for TIR-TIR interactions.Structure. 2016; 24: 437-447Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 8Avbelj M. Wolz O.O. Fekonja O. Benčina M. Repič M. Mavri J. et al.Activation of lymphoma-associated MyD88 mutations via allostery-induced TIR-domain oligomerization.Blood. 2014; 124: 3896-3904Crossref PubMed Scopus (56) Google Scholar and provide novel evidence for enhanced interaction with WT MyD88 as a plausible mechanism. Increased production of neutrophil-attracting chemokines and IL-6 likely contributes to the joint and skin phenotype, and the patient exhibited immunologic perturbations consistent with increased MyD88 signaling, such as increased leukocyte p-STAT3 levels, CD16 shedding, and increased whole-blood secretion of IL-6 and TNF-α. Interestingly, our subject lacked evidence of lymphoproliferation. It is unlikely that S222R alone is sufficient to cause lymphoproliferation because the S222R-containing SUDHL2 cell line also harbors biallelic functional deletions of A20, and its re-expression arrests proliferation.E5Compagno M. Lim W.K. Grunn A. Nandula S.V. Brahmachary M. Shen Q. et al.Mutations of multiple genes cause deregulation of NF-kappaB in diffuse large B-cell lymphoma.Nature. 2009; 459: 717-721Crossref PubMed Scopus (841) Google Scholar Similarly, ectopic expression of S209R-MyD88 (the murine ortholog of S222R) in murine B cells does not induce proliferation.E3Wang J.Q. Jeelall Y.S. Beutler B. Horikawa K. Goodnow C.C. Consequences of the recurrent MYD88 (L265P) somatic mutation for B cell tolerance.J Exp Med. 2014; 211: 413-426Crossref PubMed Scopus (67) Google Scholar Further delineating the mechanism or mechanisms by which MyD88 S222R leads to the clinical and immunologic phenotype will require additional studies, including animal modeling. All studies were completed under protocol 11-AR-0223, which was approved by the National Institute of Arthritis and Musculoskeletal and Skin Diseases/National Institute of Diabetes and Digestive and Kidney Diseases Institutional Review Board. Written informed consent and assent were obtained for conduct of research, use of photographic images, and publication of findings. Whole human exome sequencing was performed (Otogenetic, Atlanta, Ga) on the patient's, parent's, and an unaffected sister's peripheral leukocyte DNA by using Agilent 51Mb Human Exome V5 (Agilent Technologies, Santa Clara, Calif) capture and PE100-125 Illumina HiSeq2500 (Illumina, San Diego, Calif) sequencing with a 50× average read coverage. A computational pipeline was developed to process the read data and perform tasks, such as quality control, variant discovery, annotation, and filtering. Briefly, sequence reads were aligned to the human reference genome (GRC Build 37) with the Burrows-Wheeler Aligner. Burrows-Wheeler Aligner files were then processed to remove duplicate reads, refine alignment indels, and recalibrate base quality scores, according to the best practice guideline by the Genome Analysis Toolkit (GATK) from the Broad Institute. UnifiedGenotyper from GATK was used to make joint variant calls across multiple samples, followed by a variant quality score recalibration step using the GATK VQSR tool. Variants were annotated with functional effect and allele frequency in public databases and local data sets. Sex and sample kinship were analyzed to identify potential sample errors and false family relationships (ie, erroneous maternity or paternity) with KING software. Possible disease-causing mutations were selected and prioritized based on quality score, allele frequency, functional effect, inheritance, and literature review. Although no autosomal recessive mutations were detected, de novo mutations in MYD88, GFPT2, and WFIKKN2 were identified. Considering the critical role of MYD88 in the immune response and its relevance to arthritis in animal modelsE6Joosten L.A. Koenders M.I. Smeets R.L. Heuvelmans-Jacobs M. Helsen M.M. Takeda K. et al.Toll-like receptor 2 pathway drives streptococcal cell wall-induced joint inflammation: critical role of myeloid differentiation factor 88.J Immunol. 2003; 171: 6145-6153Crossref PubMed Scopus (189) Google Scholar, E7Choe J.Y. Crain B. Wu S.R. Corr M. Interleukin 1 receptor dependence of serum transferred arthritis can be circumvented by toll-like receptor 4 signaling.J Exp Med. 2003; 197: 537-542Crossref PubMed Scopus (160) Google Scholar, E9Abdollahi-Roodsaz S. van de Loo F.A. Koenders M.I. Helsen M.M. Walgreen B. van den Bersselaar L.A. et al.Destructive role of myeloid differentiation factor 88 and protective role of TRIF in interleukin-17-dependent arthritis in mice.Arthritis Rheum. 2012; 64: 1838-1847Crossref PubMed Scopus (21) Google Scholar and patients with rheumatoid arthritis,E10Elshabrawy H.A. Essani A.E. Szekanecz Z. Fox D.A. Shahrara S. TLRs, future potential therapeutic targets for RA.Autoimmun Rev. 2017; 16: 103-113Crossref PubMed Scopus (101) Google Scholar further efforts were focused on MYD88. The presence (or absence) of MYD88 S222R mutation was confirmed by means of Sanger sequencing (ACGT, Wheeling, Ill) of peripheral leukocyte DNA from all family members, as well as from isolated CD14+ monocyte, EBV-LCL, and cultured dermal fibroblast DNA from the patient and mother. Human primary dermal fibroblasts were isolated from outgrowth of Dispase (STEMCELL Technologies, Vancouver, British Columbia, Canada)–digested skin biopsy specimens and maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% FBS and 100 U/mL penicillin/streptomycin (P/S; Gibco, Carlsbad, Calif). EBV-LCLs were generated from fresh whole blood and maintained in RPMI 1640 supplemented with 20% FBS and 100 U/mL P/S. THP-1 cells were maintained in RPMI 1640 medium supplemented with 10% FBS and 100 U/mL P/S. Both U2932 and SUDHL2 cell lines were maintained in RPMI 1640 RPMI 1640 supplemented with 20% Hyclone FBS (GE Healthcare, Fairfield, Conn) and 100 U/mL P/S. All cells listed were grown at 37°C in a 5% CO2 atmosphere. Peripheral blood from the patient and control subjects was collected in sodium heparin BD Vacutainer tubes (BD, Franklin Lakes, NJ) and used within 1 hour of collection. For immunophenotyping, RBCs were lysed with BD Pharm Lyse lysing buffer and washed with PBS. The remaining cells were fixed with 4% paraformaldehyde, incubated with specific antibodies for 1 hour at 4°C, and then washed. After exclusion of dead cells with the LIVE/DEAD Fixable Red Dead Cell Stain Kit (ThermoFisher Scientific, Waltham, Mass), leukocytes were identified by using the following antibodies (BD Biosciences unless otherwise specified): anti-CD3 (SK7), anti-CD4 (SK3), anti-CD8 (SK1), anti-CCR6 (11A9), anti-CD19 (SJ25C1), anti-CD20 (2H7), anti-CD56 (B159), anti–HLA-DR (G46-6), anti-CD16 (B73.1), anti-CD123 (7G3), anti-CD11c (B-ly6), anti-CD14 (MγP9), anti-CD1c (L161; BioLegend, San Diego, Calif), anti-CD303 (201A; BioLegend), anti-CD203c (NP4D6; BioLegend), and anti-CD141 (AD5-14H12; Miltenyi Biotec, Bergisch Gladbach, Germany). For p-STAT3 analysis, peripheral blood was stained with cell subset–specific antibodies and stimulated with 50 ng/mL IL-6 (PeproTech, Rocky Hill, NJ) for 20 minutes at 37°C. Red blood cells were then lysed, and the remaining cells were fixed for 10 minutes with BD Phosflow Lyse/Fix Buffer and then permeabilized with BD Phosflow Perm Buffer III. After washing, cells were stained with anti–p-STAT3 (4/P-STAT3) overnight at 4° C. Cell-surface markers and p-STAT3 intracellular staining were measured with a BD LSR-Fortessa flow cytometer and analyzed with FlowJo software (v10.1r5; TreeStar, Ashland, Ore). For whole-blood cytokine secretion, peripheral blood from the patient and control subjects was collected in sodium heparin BD Vacutainer tubes, diluted 1:2 with RPMI 1640 medium (Gibco), and incubated for 22 hours at 37°C within 1 hour of blood collection. Supernatants were then collected and stored at −80°C. Fibroblasts (1 × 105 cells/well) from the patient and mother were seeded into 12-well plates to assess dermal fibroblast cytokine secretion. After resting overnight, cells were washed with PBS, and media were replaced. Supernatants were then collected 24 hours later and stored at −80°C. Concentrations of supernatant cytokines and chemokines from whole blood and fibroblasts were subsequently measured with a multiplex immunoassay using ProcartaPlex Human Th1/Th2 and Chemokine Panel 1 20-plex (ThermoFisher Scientific), according to the manufacturer's instructions. Neutrophil chemotaxis was measured with the CytoSelect 96-Well Cell Migration Assay (3 μm; Cell Biolabs, San Diego, Calif), according to the manufacturer's instructions. Briefly, fibroblast-conditioned supernatants from the patient and mother were generated by seeding 2.5 × 105 cells/well into 6-well plates and allowing them to rest at 37°C overnight. The next day, media were removed, and cells were washed with PBS. One milliliter of FBS-free DMEM was then placed into wells and incubated at 37°C for 24 hours. Resultant supernatants were then collected and stored at −80°C. On the day of assay, thawed supernatants were placed into feeder wells in triplicate. Freshly elutriated healthy donor neutrophils were placed into the upper chambers at a density of 5 × 105 cells/well after propidium iodide exclusion viability testing. After 4 hours, migratory cells in feeder wells were lysed, CyQuant GR Fluorescent Dye was added, and cells were quantified with a fluorescence plate reader at 480/520 nm. Four replicate 1.5-microsecond molecular dynamic calculations of the WT MyD88 TIR domain (PDB code: 4DOM, Ref. 79) or S222R mutant was performed by using Gromacs with the Charmm36 forcefield, explicit solvent, and a 1-fs time step. By using Visual Molecular Dynamics, 500 snapshots of the last 500 ns of each simulation were superposed based on the backbone atoms of the central beta sheet, and these superposed structures were clustered into 5 groups based only on the position of α-helix C (residues 243-255) backbone atoms using an rmsd distance function with 1.5 Å cutoff. For cluster analysis of α-helix C, all 4 replicates were analyzed separately. In these analyses the most populated clusters contain more than 60% of all clustered structures. THP-1–Dual KO-MyD Cells (InvivoGen, San Diego, Calif), where MYD88 gene expression is knocked out by using nuclease technology, were retrovirally transduced sequentially with N-terminal MyD88-AU1 and then C-terminal MyD88–green fluorescent protein (GFP) constructs.E11Ngo V.N. Young R.M. Schmitz R. Jhavar S. Xiao W. Lim K.H. et al.Oncogenically active MYD88 mutations in human lymphoma.Nature. 2011; 470: 115-119Crossref PubMed Scopus (1090) Google Scholar Vectors were packaged into vesicular stomatitis virus envelope (VSV-G) containing retroviral particles (Alstem, Richmond, Calif), and THP-1 cells were transduced overnight at a multiplicity of infection of 5. Successfully transduced cells were isolated by means of Ly-2 magnetic bead selection (Miltenyi Biotec), and the purities of both MyD88-AU1 and MyD88-GFP cells were verified by means of flow cytometry (Ly-2) and immunoblotting for MyD88. THP-1 clones produced for this study were MyD88 KO clones re-expressing equal amounts of WT MyD88 with 2 different tags (WT-MyD88-AU1 and WT-MyD88-GFP); WT MyD88 and S222R MyD88 with AU1 and GFP tags, respectively (WT-MyD88-AU1 and S222R-MyD88-GFP); WT MyD88 and S222R MyD88 with the alternative tags (S222R-MyD88-AU1 and WT-MyD88-GFP); and S222R MyD88 with 2 different tags (S222R-MyD88-AU1 and S222R-MyD88-GFP). THP-1–Dual KO-MyD cells contain stable integration of secreted embryonic alkaline phosphatase reporter gene containing IFN-β minimal promoter fused to 5 copies of the NF-κB consensus response element and 3 copies of the c-Rel binding site. Baseline (unstimulated) secreted embryonic alkaline phosphatase over a 24-hour period was detected by using enzyme substrate–driven colorimetry, according to the manufacturer's instructions (InvivoGen). THP-1 cells were attached to coverslips with phorbol 12-myristate 13-acetate (10 ng/mL) for 24 hours and then allowed to rest in fresh complete RPMI media for at least 72 hours. A proximity ligation assay was performed on THP-1 double-transduced clones (above) by using the Duolink In Situ Orange Mouse/Rabbit Kit (Sigma, St Louis, Mo), according to the manufacturer's instructions. Briefly, cells were washed with Hanks balanced salt solution, fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, Pa) in PBS for 15 minutes, and subsequently permeabilized/washed with 0.04% saponin (Calbiochem, San Diego, Calif). After 1 hour of blocking in 1% BSA (Sigma), 2% horse serum (Abcam, Cambridge, United Kingdom), 3% donkey goat serum (Abcam), 0.04% saponin, and 0.01% sodium azide (Sigma), cells were incubated for 16 hours at 4°C with rabbit anti-GFP antibody (ab290; Abcam) and mouse anti-AU1 (BioLegend), both at 1:1000 in blocking solution. Next, cells were washed with 0.04% saponin in PBS and incubated with anti-rabbit PLUS and anti-mouse MINUS for 1 hour. Then cells were subject to ligation for 30 minutes and amplification for 100 minutes. Cells were mounted with the kit's 4′-6-diamidino-2-phenylindole dihydrochloride–containing medium. Per cell clone, confocal images were taken from 4 randomly selected fields by using a ×63 objective, and then maximum projection images were produced from 14 z-slices. Identical imaging settings were used for all images. Proximity ligation assay events were quantitated with the CellProfiler v2.2 Spot Detection pipeline (https://github.com/tischi/cellprofiler-practical-NeuBIAS-Lisbon-2017/blob/master/practical-handout.md) from maximum projection images collected. For each experiment with each clone, at least 65 cells were counted in each of 4 fields of view or at least 260 cells for all 4 fields of view in total. Cells were lysed in buffer containing 20 mmol/L Tris-Cl (pH 7.5), 100 mmol/L NaCl, 10 mmol/L EDTA, 1% Triton X-100, protease inhibitor cocktail (Roche, Mannheim, Germany), 0.04% NaN3, 0.5 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L Na3VO4, 10 mmol/L NaF, and 10 mmol/L sodium pyrophosphate dibasic. After Bradford protein concentration measurement and normalization of protein amounts, samples were added to SDS loading buffer and heated to 95°C for 3 minutes. After PAGE (4-20% Criterion TGX; Bio-Rad Laboratories, Hercules, Calif), proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories), blocked with 5% BSA, and incubated overnight with primary antibodies at 4°C. Horseradish peroxidase–conjugated anti-rabbit IgG or anti-mouse IgG (R&D Systems, Minneapolis, Minn) secondary antibodies were used with Pierce ECL or West Pico (Thermo Fisher Scientific) to detect protein expression and visualized with Amersham Hyperfilm (GE Healthcare) or the Bio-Rad ChemiDoc Imaging System. Primary antibodies used were as follows: mouse anti-vinculin (V284; Millipore, Temecula, Calif), mouse anti-GAPDH (clone 6C5, sc-32233; Santa Cruz Biotechnology, Dallas, Tex), mouse anti–β-actin (ab8226; Abcam), rabbit, anti–β-tubulin (ab6046; Abcam), rabbit anti-MYD88 (D80F5), rabbit anti-A20/TNFAIP3 (D13H3), rabbit anti-TIRAP (ab17218; Abcam), and rabbit anti–NF-κB p65 (D14E12; all from Cell Signaling Technology, Danvers, Mass, unless otherwise noted). Quantification of immunoblots and densitometric values was performed with ImageJ software (National Institutes of Health, Bethesda, Md). SUDHL2, U2932,