1

TLR9 stimulation induces aberrant APRIL expression by tonsillar germinal center 1

B cells in IgA nephropathy 2

3

Masahiro Muto1, Benoit Manfroi2, Hitoshi Suzuki1, Kensuke Joh3, Masaaki Nagai4, 4

Sachiko Wakai5, Christian Righini6, Masayuki Maiguma1, Shozo Izui7, Yasuhiko 5

Tomino1, Bertrand Huard2* and Yusuke Suzuki1* 6

7 1Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty 8

of Medicine, Tokyo, Japan; 2University Grenoble-Alpes, INSERM, U823, Albert 9

Bonniot Institute, Grenoble, France; 3Department of Pathology, Tohoku University 10

Graduate School of Medicine, Sendai, Japan; 4Division of Nephrology and Diabetology, 11

Narita Memorial Hospital, Toyohashi, Japan; 5Department of Internal Medicine, Tokyo 12

Metropolitan Health and Medical Treatment Corporation Okubo Hospital, Tokyo, 13

Japan; 6Department of Otolaryngology–Head and Neck Surgery, Grenoble University 14

Hospital, Grenoble, France; 7Department of Pathology and Immunology, University of 15

Geneva, Geneva, Switzerland 16

* equal contribution 17

18

Correspondence should be addressed to Yusuke Suzuki and Huard Bertrand. 19

Email: yusuke@juntendo.ac.jp 20

Email: bertrand.huard@ujf-grenoble.fr 21

22

2

ABSTRACT 23

The tumor necrosis factor (TNF) family member, a proliferation-inducing ligand 24

(APRIL, TNFSF13), produced by myeloid cells, participates in the generation and 25

survival of antibody-producing plasma cells. We studied the potential role of APRIL in 26

the pathogenesis of IgA nephropathy (IgAN). We found that a significant proportion of 27

germinal centers (GC) in tonsils from IgAN patients contained cells aberrantly 28

producing APRIL, conducting to an overall upregulation of APRIL expression 29

compared to control tonsillitis patients. In IgAN GC, antigen-experienced 30

IgD-CD38+/-CD19+ B cells expressing a switched IgG/IgA BCR produced APRIL. 31

Notably, these GC B cells expressed the common cleavable APRIL-α but also the less 32

frequent uncleavable APRIL-δ and –zeta, which lacks a furin cleavage site, implying 33

expression as a membrane-bound form. Significant correlation between TLR9 and 34

APRIL expressions existed in tonsils from IgAN patients. In vitro, repeated TLR9 35

stimulation reproduced APRIL expression in tonsillar B cells from control tonsillitis 36

patients. Clinically, aberrant APRIL expression in tonsillar GC correlated with high 37

urinary protein levels, and IgAN patients harboring aberrant APRIL overexpression in 38

tonsillar GC responded well to tonsillectomy, in parallel to decreases in serum levels of 39

galactose-deficient IgA1. Taken together, our data indicate that antibody disorders in 40

IgAN are associated with TLR9 induced aberrant expression of APRIL in tonsillar GC 41

B cells. 42

43

3

Introduction 44

Immunoglobulin A nephropathy (IgAN) is the most common form of 45

glomerulonephritis (GN), accounting for 25%-50% of patients with primary GN. 46

Accumulating evidences now suggest that 30%–40% of IgAN patients progress to 47

end-stage renal disease (ESRD) within 20 years from the estimated time of disease 48

onset1, 2. The lack of comprehensive understandings of IgAN development impairs the 49

design of a specific treatment for this disease. The pathogenesis of IgAN may be 50

associated with systemic immune dysregulation from a mucosa-bone marrow axis rather 51

than an abnormality intrinsic to the renal resident cells3, 4. At the mucosal level, 52

infections, particularly in the upper respiratory tract, exacerbate clinical manifestations 53

in IgAN patients. Recent studies report that aberrantly O-glycosylated IgA1, i.e. 54

galactose-deficient IgA1 (Gd-IgA1) and the subsequently formed IgA immune 55

complexes (IC) with glycan-specific autoantibodies are pivotal to the development of 56

IgAN5-7. 57

A proliferation inducing ligand (APRIL) is a member of TNF superfamily of ligands 58

expressed as a type II transmembrane protein8. APRIL is usually cleaved in the Golgi 59

apparatus by a furin convertase, and then secreted as a soluble ligand9. Myeloid and 60

mucosal epithelial cells produced APRIL10-12. APRIL binds to two members of the TNF 61

receptor family: the B-cell maturation antigen (BCMA) and transmembrane activator 62

and calcium modulator and cyclophilin ligand interactor (TACI)13. Functionally, APRIL 63

mediates class switch, mostly for IgA10, 14. APRIL is also crucial for long-term survival 64

of plasma cells in the bone marrow and mucosa11, 12, 14-17. Recently, high serum level of 65

APRIL in IgAN patients correlating with urinary proteins was reported18, 19. In addition, 66

a genome-wide association study of IgAN patients suggested APRIL (TNFSF13) to be a 67

susceptibility gene20. However, the cellular source of APRIL production and the 68

pathway by which this molecule is upregulated in IgAN remains ill defined. 69

Toll-like receptors (TLRs) are a family of germ-line encoded receptors that 70

recognize a diverse range of conserved molecular motifs commonly found in microbial 71

pathogens. TLR9 recognizes unmethylated DNA sequences in bacterial and viral DNA, 72

and is involved in innate immune responses by providing protective immune responses 73

against invading viral and bacterial pathogens21, 22. Although TLR9 has been shown to 74

4

be implicated in the development of kidney diseases including IgAN 23-28, the 75

mechanisms by which TLR9 activation contribute to the development of IgAN are 76

poorly understood. 77

78

5

Results 79

Up-regulation of APRIL expression in tonsillar germinal center (GC) of IgAN 80

patients 81

Real-time quantitative PCR (RT qPCR) analyses in IgAN (n = 24) and chronic 82

tonsillitis (CT) (n = 6) patients revealed that tonsillar APRIL mRNA expression was 83

significantly higher in IgAN than that in CT (Figure 1A; P<0.01). 84

Immunohistochemistry with Stalk-1, an anti-APRIL antibody that detect 85

APRIL-producing cells, further showed that APRIL-producing cells were present in the 86

tonsillar epithelium and outside, in cells identified as neutrophils, of both IgAN (n=55) 87

and CT (n = 12) patients (Figure 1B), consistent with our previous report in CT 88

patients11. Stalk-1 and elastase (neutrophil specific) co-staining did not reveal a 89

difference in the number of infiltrating neutrophils between IgAN and CT patients 90

(Figure 1C). The epithelial staining by Stalk-1 was also not different. One obvious 91

difference was the presence of a substantial number of Stalk-1+ APRIL-producing cells 92

in GC in IgAN patients. We observed that the percentage of Stalk-1+GC (27.4 ± 21.3) in 93

IgAN patients was significantly higher than that in CT patients (7.2 ± 6.8; P<0.01) 94

(Figure 1D). 95

96

97

Ig-switched GC B cells produce APRIL in tonsils of IgAN patients 98

Stalk-1+ cells in tonsillar GC of IgAN patients expressed CD19, identifying them as B 99

cells (Figure 2A). We further observed that Stalk-1+ APRIL-producing cells in tonsillar 100

GC had a non-switched IgM and switched IgG or IgA phenotypes. Flow cytometric 101

analyses revealed that the Stalk-1+ CD19+ cell population had lost IgD surface 102

expression and variably expressing the GC activation marker CD38 in IgAN patients (n 103

= 13) (Figure 2B, upper left panel). We also detected this Stalk-1+ GC B-cell population 104

in CT (n = 4) (Figure 2B, lower left panel), but this cell population was clearly 105

increased in some IgAN patients (Figure 2B, right panel; P<0.05). 106

107

108

GC B cells express an uncleavable form of APRIL in IgAN patients. 109

6

The Stalk-1 staining pattern obtained in GC B cells from IgAN patients was clearly 110

different from the one in PMN cells described previously11 (Figure 3A). We additionally 111

performed immunohistochemical analysis with Aprily-2 antibody, which recognizes the 112

secreted part of APRIL29. We observed that the Aprily-2 staining colocalized with 113

Stalk-1 in tonsillar GC from IgAN patients (Figure 3B). Such colocalization of Stalk-1 114

and Aprily-2 has never been observed in several healthy and pathological tissues30. A 115

recent study reported a possible expression of APRIL-δ and -zeta, APRIL isoforms 116

lacking the consensus motif for the furin convertase, in B-cell precursor acute 117

lymphoblastic leukemia (B-ALL)31, and such isoforms could be stained with both 118

Stalk-1 and Aprily-2. RT-PCR analyses revealed that tonsillar B cells from IgAN and 119

CT patients indeed expressed not only APRIL-δ and –zeta in addition to the common 120

furin-cleavable APRIL-α (Figure 3C). RT qPCR further showed that the abundances of 121

APRIL-α and APRIL-δ/zeta mRNAs in tonsillar B cells of IgAN patients were 122

significantly higher than those in CT patients (Figure 3D). 123

124

125

TLR9 stimulation induces APRIL expression in GC B cells 126

Levels of TLR9 mRNA in whole tonsils and tonsillar B cells of IgAN patients were 127

significantly higher than those of CT patients (Figure 4A), and an increase of TLR9 128

mRNA well correlated with an increase of APRIL-α and δ/zeta mRNA in tonsillar B 129

cells of IgAN patients (Figure 4B). 130

We next stimulated whole tonsillar cells from CT patients with the TLR9 ligand 131

CpG-oligodeoxynucleotides (CpG-ODN), and analyzed APRIL expression on CD19+ B 132

cells. A daily stimulation induced a reactivity of CD19+ cells with Stalk-1 and Aprily-2 133

antibodies, starting at day 3, with a maximum seen at day 7, in CD19+ cells. (Figure 5A). 134

The APRIL reactivity was observed intracellularly with a limited signal at the cells 135

surface. The weak surface APRIL expression on CpG-stimulated B cells was consistent 136

with the absence of surface staining observed ex vivo. Repeated TLR9 stimulation 137

weakly but clearly upregulated expression of the APRIL receptors TACI and BCMA as 138

well (Figure 5B). Consistent with analyses in Figure 5A, same APRIL reactivities were 139

observed in studies with purified tonsillar B cells at day 7 (Figure 5C). Taken together, 140

7

these indicate a putative autocrine APRIL signaling in CpG-stimulated B cells. 141

142

143

APRIL expression in tonsillar GC is associated with the severity of IgAN and 144

treatment responses to tonsillectomy 145

We assessed whether the extent of Stalk-1+GC in tonsils may affect the severity of 146

IgAN and therapeutic responses to tonsillectomy. The percentage of Stalk-1+GC was 147

significantly higher in IgAN patients with an elevated proteinuria (Figure 6A; P<0.01). 148

Figure 1D showed that even CT patients showed Stalk-1+GC, but less than 10%, 149

suggesting that Stalk-1+GC≥10% in tonsils may be more characteristic to IgAN. 150

Therefore, we next focused on IgAN patients with Stalk-1+GC more than 10%. Urinary 151

protein level in IgAN patients with Stalk-1+GC≥10% was significantly higher than that 152

in IgAN patients with Stalk-1+GC<10% (Figure 6B; P<0.05). Percentage of IgAN 153

patients whose proteinuria decreased more than 50% after the tonsillectomy alone was 154

significantly higher in patients with Stalk-1+GC≥10% (62.8%) than those with 155

Stalk-1+GC<10% (25.0%; P=0.0201) (Table 2). Notably, the efficacy of tonsillectomy 156

was similarly observed in severe cases of IgAN who showed proteinuria more than 157

0.5g/gCr (P=0.048), independently of basal levels of proteinuria before the treatment 158

(Supplement Table 1). Change of serum levels of whole IgA and Gd-IgA1 before and 159

after tonsillectomy was then evaluated. Significantly higher decrease of serum Gd-IgA1 160

(P<0.05), but not whole IgA, was observed in IgAN patients with Stalk-1+GC≥10% 161

(Figure 6C and 6D). We also compared proteinuria levels before and after tonsillectomy 162

in whole IgAN patients (Figure 6E), those with Stalk-1+GC ≥ 10% (Figure 6F) and 163

those with Stalk-1+GC<10% (Figure 6G). There were significant differences between 164

before and after tonsillectomy in the preceding two groups (*P<0.01), despite no 165

significance in IgAN patients with Stalk-1+GC<10%. 166

167

8

Discussion 168

Mucosal immune dysregulation has already been reported in the pathogenesis of IgAN. 169

However, the underlying mechanism remains unclear. APRIL and B cell activating 170

factor belonging to the tumor necrosis factor family (BAFF) derived from myeloid cells 171

such as neutrophils, monocytes and dendritic cells are TNF super family members best 172

known for their roles in the survival and maturation of B cells. Recent studies revealed 173

that mature B-cell neoplasms, including chronic lymphocytic leukemia32, follicular 174

lymphoma and diffuse large B cell lymphoma may start to produce APRIL33, 34 by 175

themselves. This aberrant production of APRIL was also observed in B cells from 176

patients suffering from autoimmune diseases such as systemic lupus erythematous 177

(SLE)35. In fact, repeated stimulation of B cells induced their expression of APRIL36. 178

The present study shows, for the first time, an aberrant up-regulation of APRIL in 179

tonsillar GC B cells from IgAN patients. 180

In GC B cells from IgAN, we observed that the reactivity of the two APRIL-specific 181

antibodies, Stalk-1 and Aprily-2, was clearly different from the one obtained in other 182

organs. Indeed, we observed an uncommon co-staining of Stalk-1 and Aprily-2 183

revealing the presence of a full-length form of APRIL. The detection of the two furin 184

uncleavable isoforms of APRIL, δ and zeta mRNAs, is consistent with this observation. 185

This uncleavable full-length APRIL was detected intracellularly, stored most likely in 186

vesicles warranting further investigations (see Figure 3A). 187

Exacerbation of IgAN upon upper respiratory infections allows speculation on the 188

participation of exogenous antigens in disease progression. The palatine tonsils have a 189

unique cellular composition in the reticulated subepithelium, which is ideal for 190

productive antigen sampling for rapid and broad defense against microorganisms at the 191

gate of the respiratory and digestive tracts. Transient mucosal activation of a pattern 192

recognition receptor, such as TLR, by pathogen-associated molecular patterns in 193

IgAN-prone mice is sufficient to exacerbate this disease, with rapid serum elevation of 194

IgA and ICs23. We recently demonstrated that tonsillar levels of TLR9 expression, but 195

not that of other TLRs, were associated with the disease activity of IgAN and clinical 196

outcome of tonsillectomy24-28. Furthermore, the TLR9 genotype was strongly associated 197

with histological severity of IgAN23. Genome-wide scan identifies a copy number 198

9

variable region at 3p21.1 that influences the TLR9 expression levels in IgA nephropathy 199

patients.37 Accordingly, these findings suggest that tonsillar TLR9 signaling pathways 200

may be involved in the pathogenesis of human IgAN. TLR9-ligand CpG-ODN 201

increased the expressions of the APRIL receptors BCMA and TACI on B cells, and 202

enhance B-cell activation and Ig secretion38, 39. We are reporting here that chronic 203

CpG-ODN stimulation induced APRIL production by tonsillar B cells. The findings of 204

the present study provide a rationale of tonsillectomy and indicate that TLR9-APRIL 205

axis is a promising specific target for the future treatment, apart from non-specific 206

immunosuppressants or tonsillectomy. Besides, although the possible contribution of 207

specific exogenous antigens to the pathogenesis of IgAN including Haemophilus 208

parainfluenzae has been discussed40, there are no consistent antigens yet. Our previous 209

and recent studies regarding the involvement of TLR9 in the pathogenesis of human and 210

murine IgAN indicate that specific antigens are not required for the development of 211

IgAN. However, it seems that there is a biased bacterial flora in tonsil of human IgAN41, 212

suggesting that actual exogenous antigens in the pathogenesis may be limited. 213

We observed that tonsillar levels of APRIL correlated with disease activity and 214

treatment responses to tonsillectomy, indicating that tonsillar GC B cells may be 215

involved in the pathogenesis via their production of APRIL. It is now widely accepted 216

that Gd-IgA1 and related IC are essential effector molecules to induce glomerular 217

damages in IgAN7, 42. Serum levels of these molecules, indeed, have clinical diagnostic 218

potential for the assessment of prognosis and disease activity for IgAN, independent of 219

information from renal biopsy42-45. Reports showed abnormal glycosylation of tonsillar 220

IgA46, 47 and aberrant cytokine profiles in tonsillar B cells, leading to the 221

underglycosylation of IgA1 in IgAN patients48-51. Because total IgA is decreased by 222

approximately 10 % on an average after tonsillectomy alone in IgAN patients27, and 223

patients who showed a large decrease of serum IgA after the tonsillectomy had better 224

clinical outcome, the palatine tonsil was hypothesized to be a major delivery sources of 225

nephritogenic IgA27. Indeed, we recently showed that Gd-IgA1 was significantly 226

decreased after tonsillectomy in IgAN patients who also showed a significant 227

improvement in urinalysis just after tonsillectomy26. Present study further demonstrated 228

that IgAN patients with abundant expression of Stalk-1 in tonsillar GC showed more 229

10

proteinuria and better clinical outcomes after the tonsillectomy including improvement 230

of proteinuria and decrease of serum Gd-IgA1 levels. These findings suggest that 231

palatine tonsils with overexpression of APRIL may be one of the major delivery source 232

of nephritogenic IgA. However, some patients notably showed improved hematuria and 233

serum GdIgA1 levels following steroid pulse therapy after the tonsillectomy as 234

compared with just after tonsillectomy, suggesting that GdIgA1-producing cells may 235

also be localized outside the tonsils26. Recent data have revealed that some of the 236

NALT-derived and activated B cells, and even tonsillar B cells, can migrate from 237

inductive mucosal sites to systemic effector sites including bone marrow through 238

guiding adhesion molecules and chemokine/chemokine receptors52, 53. In addition, our 239

recent study revealed that IgA1 secreted by Epstein–Barr virus (EBV)-immortalized B 240

cells from the peripheral blood of IgAN patients was mostly polymeric with 241

galactose-deficient sialylated O-glycans54. These findings support the hypothesis that 242

GdIgA1-producing B cells may travel between the tonsils and systemic lymphoid 243

organs and produce the nephritogenic IgA outside of mucosal sites. Moreover, bone 244

marrow transplantation with an IgAN donor reconstitutes IgAN in humans and mice55, 56, 245

suggesting that GdIgA1-producing B cells could localize in the bone marrow. Indeed, it 246

has been reported that IgA plasma cells containing subclass IgA1 were increased in 247

IgAN patients compared to controls57, 58. It has also been shown that there was an 248

increase in the proportion of IgA+ cells which express J chain mRNA, which is essential 249

for the production of dimeric IgA, in the bone marrow of IgAN patients59. These 250

evidences emphasize the possibility that the bone marrow could be the production site 251

of the IgAl found in the circulation and mesangial deposits in IgAN patients. Thus, we 252

can speculate that mucosally-primed/activated GdIgA1-producing B cells are 253

disseminated to systemic organs such as lymph nodes and tonsils, or the bone marrow. 254

In conclusion, APRIL+ GC B cells in tonsils may determine the disease activity of 255

IgAN patients, presumably via production of Gd-IgA1 prior to IC formation. Aberrant 256

TLR9 activation in tonsillar B cells may be involved in the underlying mechanisms of 257

the tonsillar overexpression of APRIL, partly with APRIL variants, in IgAN (Figure 7). 258

259

11

Methods 260

Patients and treatment protocol 261

Fifty-five biopsy-proven IgAN (26 males) and 12 CT (6 males) patients, who had 262

undergone tonsillectomy at the Department of Otorhinolaryngology of Juntendo 263

University Hospital, Narita Memorial Hospital and Tokyo Metropolitan Health and 264

Medical Treatment Corporation Okubo Hospital, were included in this study. Patient 265

demographics and clinical characteristics are summarized in Table 1. Both before and 266

after tonsillectomy, IgAN patients were evaluated for the following clinical outcomes: 267

proteinuria [g/g creatinine (Cr); g/gCr] and serum levels of IgA and Gd-IgA1. The 268

average duration from tonsillectomy to quantification of these clinical parameters was 269

69.2±47.2 days. This study was conducted in accordance with the principles of the 270

Declaration of Helsinki and the study protocol approved by the Institutional Review 271

Boards of each hospital. Informed consents were obtained from all patients before 272

inclusion in the study. 273

274

275

Real-time quantitative reverse transcription PCR and sequencing 276

qPCR on RNA isolated from tonsillar tissues and cells was performed as described 277

previously27. A Homo sapiens-specific Taqman gene expression assay (Life 278

Technologies Co, Carlsbad, CA) was purchased for the TNF (ligand) superfamily, 279

member 13 (APRIL) (Hs00601664_g1) and TLR9 (Hs00370913_s1) as well as for an 280

endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 281

(Hs99999905_m1). 282

A SYBR Green PCR Master Mix (Applied Biosystems, Foster city, CA) and a 7500 283

Real-Time PCR System (Applied Biosystems) were also used for qPCR for APRIL-α 284

and δ/zeta expression. Transcript levels were normalized by GAPDH. The following 285

primers synthesized by Life Technologies Co were used: APRIL-α, 286

5’-AGGAGAGCAGTGCTCACCC-3’ and 5’-CTCCAGCATCCTGGATTCGG-3’; 287

APRIL-δ, 5’-AGAGTCTCCCGGAGCAGCAG-3’ and 288

5’-CTCCAGCATCCTGGATTCGG-3’; and GAPDH, 289

5’-TGACTCCGACCTTCACCTTC-3’ and 5’-CTCTGCTCCTCCTGTTCGAC-3’. 290

12

For sequencing purposes, total RNA from tonsillar B cells was extracted using the 291

QIA shredder (Qiagen, Valencia, CA) and the RNeasy Mini Kit (Qiagen). The PCR was 292

performed with Takara Ex Taq DNA polymerase (Takara, Shiga, Japan). PCR 293

fragments were isolated from 2% agarose gels using a gel extraction kit (Qiagen). PCR 294

products were ligated into the pMD20 vector (Takara). All standard cloning and 295

plasmid propagation was performed in Escherichia coli strain INVαF’ (Invitrogen AG, 296

Basel, Switzerland). Clones were first screened by restriction enzyme digestion (EcoRI 297

and SphI, Takara) and subsequent RT-PCR were subjected to sequence analysis with 298

primers Seq Forward M13 Primer RV (5’-CAGGAAACAGCTATGAC-3’) and Seq 299

Reverse M13 Primer M4 (5’-GTTTTCCCAGTCACGAC-3’). The REH cell line was 300

obtained from the American Tissue and Cell Collection (Manassas, VA). 301

302

303

Immunohistochemical analysis 304

For immunohistochemistry, the paraformaldehyde-fixed paraffin-embedded tonsillar 305

samples from IgAN and CT patients were cut at a thickness of 3 μm. All sections were 306

deparaffinized in xylene followed by 100% ethanol, and then placed in a freshly 307

prepared methanol/0.3% H2O2 solution for 10 min. Microwave antigen retrieval was 308

performed with a hot 0.01 mol/L citrate buffer for 20 min. The sections were cooled to 309

room temperature, and then blocked with a blocking solution (DS Pharma Biomedical, 310

Osaka, Japan). The primary antibody Stalk-1 was used at 5µg/ml. The polyclonal rabbit 311

antiserum Stalk-1 was raised against a peptide in the membrane-proximal part of 312

APRIL extracellular domain remaining associated to the cell membrane after furin 313

cleavage 29. The secondary antibody was a horseradish peroxidase (HRP)-labeled 314

anti-rabbit antibody (1:50, Dako, Tokyo, Japan). Sections were washed with 315

phosphate-buffered saline (PBS; pH 7.4) three times after each incubation. An 316

individual GC was considered a Stalk-1＋ GC when 25% of its area was covered by 317

Stalk-1+ cells. Image acquisition was performed with a ×40 objective. Cell numeration 318

was performed in a tissue area of 30mm2. Stainings were evaluated by two 319

nephrologists who were blinded to patients’ clinical data. 320

For immunofluorescence staining, tonsillar tissues were mounted in optimal cutting 321

13

temperature (OCT) compound (Sakura Finetek, Tokyo, Japan), immersed in liquid 322

nitrogen, and stored at -80°C. These frozen specimens were cut into 3 mm sections and 323

fixed with 4% paraformaldehyde at -20°C for 10 min. Paraformaldehyde-fixed frozen 324

tonsillar tissue sections were then stained with Stalk-1, Aprily-2 (mouse IgG, 2 µg/ml), 325

anti-elastase (NP57, mouse IgG, 1:200, Dako), anti-CD19 (LE-CD19, mouse IgG, 326

1:100, Dako), anti-human IgG (3E8, mouse IgG, 0.5 µg/ml, Santa Cruz, Dallas, TX), 327

anti-human IgA (47C12, mouse IgG, 1:200, Santa Cruz), and anti-human IgM (R1/69, 328

mouse IgG, 1 µg/ml, Santa Cruz). The mAb Aprily-2 was raised against the C-terminal 329

TNF homology domain of APRIL, secreted on furin cleavage 29. Slides were incubated 330

with the following secondary reagents: Alexa 488-conjugated anti-rabbit Ig (Invitrogen 331

AG) and Alexa 555-conjugated anti-mouse Ig (Invitrogen AG). Nuclei were visualized 332

with 4′,6-diamidine-2′phenylindole dihydrochloride (DAPI; Boehringer Mannheim, 333

Indianapolis, IN). Images were acquired with a confocal laser-scanning microscope 334

(Fluoview FV1000, Olympus, Tokyo, Japan). The number of Stalk-1+ elastase+ positive 335

cells was quantified with KS400 Image Analysis System (Carl Zeiss, Oberkochen, 336

Germany) by two nephrologists. 337

338

339

ELISA for Gd-IgA1 340

The serum level of Gd-IgA1 was measured by lectin ELISA using GalNAc-specific 341

lectin from Helix aspersa (HAA; Sigma, St. Louis, MO) as previously reported45, 54, 60, 61. 342

Diluted sera were added 100 ng per well of serum IgA. The captured IgA was treated 343

with 10 mU/ml neuraminidase (Roche Diagnostics Corp. Indianapolis, IN) to remove 344

terminal sialic acid residues54, 60. The desialylated IgA1 was then reacted with 345

biotin-labeled HAA and subsequently developed absorbance was measured at 490 nm. 346

The HAA reactivity of IgA1 in each sample was then calculated as OD units/100 ng of 347

serum IgA. Naturally galactose-deficient IgA1 (Ale) myeloma protein60 treated with 348

neuraminidase and was used as the standard. Serum level of total Gd-IgA1 was 349

expressed in relative Units, calculated by multiplying the normalized HAA reactivity by 350

the amount of IgA in the serum sample (mg/ml). 351

352

14

353

Tonsillar cell preparation 354

After surgery, tonsil samples were dissected into small pieces in 2 mg/mL collagenase 355

IV (Worthington Biochemical Corporation, Lakewood, NJ), and filtered on a 100 μm 356

cell strainer. Tonsil cell stimulation was performed daily with 10 µg/ml of CpG ODN 357

1826 and control ODN 1982 (Microsynth, Balgach, Switzerland). Tonsillar B cells were 358

purified using a Dynabeads untouched Human B cells Kit (Invitrogen AG) according to 359

the manufacturer’s instructions. 360

361

362

Flow cytometric analysis 363

1×106 tonsillar cells were stained for flow cytometry. The cells were preincubated with 364

Fc receptor blocking reagent (MBL, Aichi, Japan), and incubated for 30 min at 4°C with 365

fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD19 antibody 366

(Biolegend, San Diego, CA), APC mouse anti-human CD38 (BD Pharmingen, San Jose, 367

CA), PE mouse anti-human IgD (BD Pharmingen). Biotinylated anti-TACI and BCMA 368

have been previously described62. PE-conjugated streptavidin was from BD Pharmingen. 369

Total staining was performed after permeabilization with a Cytofix/Cytoperm solution 370

(BD Pharmingen) for 30 min at 4°C. After washing twice in Perm/Wash solution, cells 371

were incubated for 30 min at 4°C with Stalk-1 (5 μg/ml) and Aprily-2 (5 µg/ml). 372

Brilliant Violet 421 donkey anti-rabbit or mouse IgG (Biolegend) was used as 373

secondary antibodies. After washing the cells twice in BD Perm/Wash solution, labeled 374

cells were analyzed by flow cytometry using a FACSVerse flow cytometer (Becton 375

Dickinson, Franklin Lakes, NJ) and the FlowJo program (Tree Star, Ashland, OR). 376

377

378

Statistical Analysis 379

Statistical analyses were performed using Graph Pad PRISM software, version 6.0 380

(GraphPad, La Jolla, CA). Comparisons between groups were analyzed by the 381

Mann-Whitney U test. Spearman’s regression analysis was used to analyze the 382

correlation between two variables. Differences at P<0.05 were considered significant. 383

15

384

385

Acknowledgements 386

The authors thank Drs. Takaaki Oobayashi, Kanae Yoshikawa, and Hiroshi Kitagawa for 387

preparation of tonsillar samples. Excellent technical assistance was provided by Ms. 388

Terumi Shibata, Drs. Masao Kihara, Akemi Kawasaki, Takako Ikegami and Tomomi 389

Ikeda (Division of Molecular and Biochemical Research, Juntendo University Graduate 390

School of Medicine). This study was supported by grants from the Japanese Swiss 391

Science and Technology Cooperation Program, the Strategic International Research 392

Cooperative Program, the Japan Science and Technology Agency (BH, YT and YS), by 393

the foundation Finovi (BH), INSERM (BH), and Joseph Fourier University (BH), and 394

by Grant-in-Aids from the Ministry of Health, Labor and Welfare of Japan (MM, HS, 395

MM, YS). 396

397

398

Authorship 399

Contribution: M.M. H.S. B.M. and Y.S. performed the experiments and analyzed the 400

data. Y.S. S.I. and B.H. designed the study. Y.S., M.M., S.I. and B.H. wrote the paper. 401

K.J., M.N., S.W., C.R., and M.Maiguma provided patients materials and information. 402

Y.S. and Y.T. designed the research protocol. 403

404

405

DISCLOSURES 406

None. 407

16

Figure 1 408

Increased APRIL expression in tonsils from IgAN patients. 409

A) Tonsillar APRIL mRNA expressions in IgAN patients (n = 24) were significantly 410

higher than those in chronic tonsillitis patients (CT; n = 6) (*P<0.01). Bars represent the 411

mean ± SEM. B) Immunohistochemistry with Stalk-1 (specific for APRIL-producing 412

cells) in IgAN (upper panels) and CT (lower panels) patients. Representative germinal 413

centers (GC) are shown on the right panels. Scale bars=250µm and 100µm for left and 414

right panels, respectively. Arrow and arrowhead mark a Stalk-1 stained neutrophil and 415

epithelial cells, respectively. Pictures shown are representative of 56 tonsils from IgAN 416

patients and 12 tonsils from CT patients. C) Quantification of Stalk-1+ elastase+ 417

neutrophils showed no significant difference. D) However, percentage of GC containing 418

APRIL-producing cells (Stalk-1+GC) was significantly different in total tonsillar GC 419

from IgAN patients and CT patients (*P<0.01). 420

421

422

Figure 2 423

CD19+ B cells produce APRIL in tonsillar GC of IgAN patients. 424

A) IgAN tonsils showed co-staining for Stalk-1 (green) and CD19, IgM, IgG and IgA 425

(red). A representative GC is shown. Pictures shown are representative of tonsils from 426

IgAN patients. Scale bars = 20µm. B) A cell suspension from IgAN and CT tonsils was 427

surface stained for CD19, CD38, IgD and after cell permeabilization for Stalk-1 (left 428

panel). Plots for cells gated on CD19 are representative of 13 IgAN and 4 CT patients. 429

The percentage of Stalk-1+ cells among CD19＋IgD－CD38+/- cells is also shown (right 430

panel) (**P<0.05). 431

432

433

Figure 3 434

Tonsillar GC B cells of IgAN express cleavable and uncleavable APRIL. 435

A) IgAN tonsils were stained for Stalk-1. A representative GC B cells (right panel) are 436

shown. Pictures shown are representative of 56 IgAN patients. B) IgAN tonsils were 437

co-stained for Stalk-1 (green) and Aprily-2 (red). A representative GC is shown. Scale 438

17

bars = 20µm. C) Predicted amino-acid sequences of different isoforms of APRIL. The 439

GenBank accession numbers for APRIL-α, APRIL-δ and APRIL-zeta are NM_003808, 440

NM_001198622 and NM_001198623.1, respectively. The furin cleavable site lacking in 441

APRIL-δ and APRIL-zeta is highlighted in grey. Identities are indicated by dashes and 442

deletions by dots. Numbers indicate amino-acid positions. D) Correlation between 443

APRIL-α and APRIL-δ/zeta mRNA expression in purified tonsillar B cells from IgAN 444

(n = 20) and CT (n = 6) patients. Both APRIL-α and APRIL-δ/zeta mRNA expression 445

in tonsillar B cells were significantly higher in IgAN patients (**P<0.05). Bars 446

represent the mean ± SEM. 447

448

449

Figure 4 450

Correlation between TLR9 and APRIL mRNA expression in IgAN patients 451

A) TLR9 mRNA expressions in whole tonsils (*P<0.01) (left panel) and purified 452

tonsillar B cells (right panel) (**P<0.05) were significantly higher in IgAN. Bars 453

represent the mean ± SEM. B) TLR9 and APRIL-α (left panel) or δ/zeta (right panel) 454

mRNA expressions in tonsillar B cells were well correlated in IgAN patients. 455

456

457

Figure 5 458

TLR9 activation induces APRIL expression in tonsillar B cells. 459

A) Tonsillar B cells isolated from CT patients were stimulated daily with 10 µg/ml of 460

CpG. APRIL expression is shown on viable (upper panel) and permeabilized (middle 461

panel) gated CD19+ B cells. B) Surface expressions of TACI and BCMA are also shown 462

(lower panel). C) CD19+ B cells from CT patients were purified on a FACS ARIA (BD 463

Pharmingen) by positive selection. Purified CD19+ B cells were stimulated daily with 464

10 µg/ml of CpG. APRIL expression is shown on permeabilized cells. Shaded 465

histograms represent control isotype-matched reactivity. Dotted and straight lines 466

represent indicated antibody reactivities on control and CpG ODN stimulated cells, 467

respectively, at day 7. Histogram plots are representative of at least three experiments 468

performed with tonsils from independent patients. 469

18

470

471

Figure 6 472

Correlation between APRIL expression in tonsillar GC and disease activity in 473

IgAN patients. 474

A) Percentage of Stalk-1+GC in tonsils of IgAN patients and their proteinuria level. The 475

percentage of Stalk-1+GC in IgAN patients with proteinuria more than 1g/gCr was 476

significantly higher than that in those less than 1g/gCr (*P<0.01). B-D) Comparison of 477

proteinuria level (B), % decrease of the serum IgA (C) and the serum Gd-IgA1 levels 478

(D) between IgAN patients with the percentage of Stalk-1+GC ≥ 10%, and those with 479

the percentage of Stalk-1+GC < 10%. IgAN patients with Stalk-1+GC ≥ 10% showed 480

significantly higher proteinuria before tonsillectomy (**P<0.05) and larger decrease of 481

serum levels of Gd-IgA1 after tonsillectomy (**P<0.05) than those with Stalk-1+GC < 482

10%. E-G) Comparison of proteinuria levels before and after tonsillectomy in whole 483

IgAN patients (E), those with Stalk-1+GC ≥ 10% (F) and those with Stalk-1+GC<10% 484

(G). There were significant differences between before and after tonsillectomy in the 485

preceding two groups (*P<0.01), despite no significance in IgAN patients with 486

Stalk-1+GC<10%. The average duration from tonsillectomy to quantification of these 487

clinical parameters was 69.2±47.2 days. 488

489

490

Figure 7 491

Cross-talk between APRIL and TLR9 on B cells in tonsillar GC of IgAN patients. 492

Present study revealed aberrant APRIL expression in tonsillar GC B cells from IgAN 493

patients. Based on our findings, we hypothesize that activation of intracellular TLR9 494

through exogenous antigens may be involved in this overexpression consisting not only 495

APRIL-α but also uncleaved APRIL, such as APRIL-δ/zeta in tonsillar GC B cells of 496

IgAN patients. This TLR9 activation also upregulates expression of TACI and BCMA, 497

and increases both BCR signaling and APRIL sensitivity. This aberrant APRIL 498

expression may induce long-term survival of GC B cells responsible for the production 499

of aberrant antibodies including Gd-IgA1, and thereby, contribute to subsequent 500

19

progression of IgAN. 501

502

20

References 503

1. Barratt J, & Feehally J: IgA nephropathy. J Am Soc Nephrol 16: 2088-2097, 2005 504

2. Appel GB, & Waldman M: The IgA nephropathy treatment dilemma. Kidney Int 69: 505

1939-1944, 2006 506

3. Suzuki Y, Suzuki H, Sato D, Kajiyama T, Okazaki K, Hashimoto A, Kihara M, 507

Yamaji K, Satake K, Nakata J, Aizawa M, Novak J, Tomino Y: Reevaluation of the 508

mucosa-bone marrow axis in IgA nephropathy with animal models. Adv 509

Otorhinolaryngol 72: 64-67, 2011 510

4. Suzuki Y, & Tomino Y: Potential immunopathogenic role of the mucosa-bone 511

marrow axis in IgA nephropathy: Insights from animal models. Semin Nephrol 28: 512

66-77, 2008 513

5. Kokubo T, Hiki Y, Iwase H, Horii A, Tanaka A, Nishikido J, Hotta K, Kobayashi Y: 514

Evidence for involvement of IgA1 hinge glycopeptide in the IgA1-IgA1 interaction 515

in IgA nephropathy. J Am Soc Nephrol 8: 915-919, 1997 516

21

6. Tomana M, Matousovic K, Julian BA, Radl J, Konecny K, Mestecky J: 517

Galactose-deficient IgA1 in sera of IgA nephropathy patients is present in 518

complexes with IgG. Kidney Int 52: 509-516, 1997 519

7. Suzuki H, Kiryluk K, Novak J, Moldoveanu Z, Herr AB, Renfrow MB, Wyatt RJ, 520

Scolari F, Mestecky J, Gharavi AG, Julian BA: The pathophysiology of IgA 521

nephropathy. J Am Soc Nephrol 22: 1795-1803, 2011 522

8. Hahne M, Kataoka T, Schroter M, Hofmann K, Irmler M, Bodmer JL, Schneider P, 523

Bornand T, Holler N, French LE, Sordat B, Rimoldi D, Tschopp J: APRIL, a new 524

ligand of the tumor necrosis factor family, stimulates tumor cell growth. J Exp Med 525

188: 1185-1190, 1998 526

9. Lopez-Fraga M, Fernandez R, Albar JP, Hahne M: Biologically active APRIL is 527

secreted following intracellular processing in the golgi apparatus by furin 528

convertase. EMBO Rep 2: 945-951, 2001 529

10. He B, Xu W, Santini PA, Polydorides AD, Chiu A, Estrella J, Shan M, Chadburn A, 530

Villanacci V, Plebani A, Knowles DM, Rescigno M, Cerutti A: Intestinal bacteria 531

trigger T cell-independent immunoglobulin A(2) class switching by inducing 532

epithelial-cell secretion of the cytokine APRIL. Immunity 26: 812-826, 2007 533

22

11. Huard B, McKee T, Bosshard C, Durual S, Matthes T, Myit S, Donze O, Frossard C, 534

Chizzolini C, Favre C, Zubler R, Guyot JP, Schneider P, Roosnek E: APRIL 535

secreted by neutrophils binds to heparan sulfate proteoglycans to create plasma cell 536

niches in human mucosa. J Clin Invest 118: 2887-2895, 2008 537

12. Matthes T, Dunand-Sauthier I, Santiago-Raber ML, Krause KH, Donze O, Passweg 538

J, McKee T, Huard B: Production of the plasma-cell survival factor a 539

proliferation-inducing ligand (APRIL) peaks in myeloid precursor cells from 540

human bone marrow. Blood 118: 1838-1844, 2011 541

13. Yu G, Boone T, Delaney J, Hawkins N, Kelley M, Ramakrishnan M, McCabe S, 542

Qiu WR, Kornuc M, Xia XZ, Guo J, Stolina M, Boyle WJ, Sarosi I, Hsu H, Senaldi 543

G, Theill LE: APRIL and TALL-I and receptors BCMA and TACI: System for 544

regulating humoral immunity. Nat Immunol 1: 252-256, 2000 545

14. Castigli E, Scott S, Dedeoglu F, Bryce P, Jabara H, Bhan AK, Mizoguchi E, Geha 546

RS: Impaired IgA class switching in APRIL-deficient mice. Proc Natl Acad Sci U 547

S A 101: 3903-3908, 2004 548

15. Belnoue E, Pihlgren M, McGaha TL, Tougne C, Rochat AF, Bossen C, Schneider P, 549

Huard B, Lambert PH, Siegrist CA: APRIL is critical for plasmablast survival in 550

23

the bone marrow and poorly expressed by early-life bone marrow stromal cells. 551

Blood 111: 2755-2764, 2008 552

16. Chu VT, Beller A, Rausch S, Strandmark J, Zanker M, Arbach O, Kruglov A, Berek 553

C: Eosinophils promote generation and maintenance of 554

immunoglobulin-A-expressing plasma cells and contribute to gut immune 555

homeostasis. Immunity 40: 582-593, 2014 556

17. Chu VT, Frohlich A, Steinhauser G, Scheel T, Roch T, Fillatreau S, Lee JJ, Lohning 557

M, Berek C: Eosinophils are required for the maintenance of plasma cells in the 558

bone marrow. Nat Immunol 12: 151-159, 2011 559

18. McCarthy DD, Kujawa J, Wilson C, Papandile A, Poreci U, Porfilio EA, Ward L, 560

Lawson MA, Macpherson AJ, McCoy KD, Pei Y, Novak L, Lee JY, Julian BA, 561

Novak J, Ranger A, Gommerman JL, Browning JL: Mice overexpressing BAFF 562

develop a commensal flora-dependent, IgA-associated nephropathy. J Clin Invest 563

121: 3991-4002, 2011 564

19. Han SS, Yang SH, Choi M, Kim HR, Kim K, Lee S, Moon KC, Kim JY, Lee H, Lee 565

JP, Jung JY, Kim S, Joo KW, Lim CS, Kang SW, Kim YS, Kim DK: The role of 566

24

TNF superfamily member 13 in the progression of IgA nephropathy. J Am Soc 567

Nephrol 2016 568

20. Yu XQ, Li M, Zhang H, Low HQ, Wei X, Wang JQ, Sun LD, Sim KS, Li Y, Foo 569

JN, Wang W, Li ZJ, Yin XY, Tang XQ, Fan L, Chen J, Li RS, Wan JX, Liu ZS, 570

Lou TQ, Zhu L, Huang XJ, Zhang XJ, Liu ZH, Liu JJ: A genome-wide association 571

study in han chinese identifies multiple susceptibility loci for IgA nephropathy. Nat 572

Genet 44: 178-182, 2011 573

21. Krieg AM: Therapeutic potential of toll-like receptor 9 activation. Nat Rev Drug 574

Discov 5: 471-484, 2006 575

22. Goodnow CC: Immunology. discriminating microbe from self suffers a double toll. 576

Science 312: 1606-1608, 2006 577

23. Suzuki H, Suzuki Y, Narita I, Aizawa M, Kihara M, Yamanaka T, Kanou T, 578

Tsukaguchi H, Novak J, Horikoshi S, Tomino Y: Toll-like receptor 9 affects 579

severity of IgA nephropathy. J Am Soc Nephrol 19: 2384-2395, 2008 580

25

24. Kajiyama T, Suzuki Y, Kihara M, Suzuki H, Horikoshi S, Tomino Y: Different 581

pathological roles of toll-like receptor 9 on mucosal B cells and dendritic cells in 582

murine IgA nephropathy. Clin Dev Immunol 2011: 819646, 2011 583

25. Maiguma M, Suzuki Y, Suzuki H, Okazaki K, Aizawa M, Muto M, Tomino Y: 584

Dietary zinc is a key environmental modifier in the progression of IgA nephropathy. 585

PLoS One 9: e90558, 2014 586

26. Nakata J, Suzuki Y, Suzuki H, Sato D, Kano T, Yanagawa H, Matsuzaki K, 587

Horikoshi S, Novak J, Tomino Y: Changes in nephritogenic serum 588

galactose-deficient IgA1 in IgA nephropathy following tonsillectomy and steroid 589

therapy. PLoS One 9: e89707, 2014 590

27. Sato D, Suzuki Y, Kano T, Suzuki H, Matsuoka J, Yokoi H, Horikoshi S, Ikeda K, 591

Tomino Y: Tonsillar TLR9 expression and efficacy of tonsillectomy with steroid 592

pulse therapy in IgA nephropathy patients. Nephrol Dial Transplant 27: 1090-1097, 593

2012 594

28. Watanabe T, Kanamaru Y, Liu C, Suzuki Y, Tada N, Okumura K, Horikoshi S, 595

Tomino Y: Negative regulation of inflammatory responses by immunoglobulin A 596

26

receptor (FcalphaRI) inhibits the development of toll-like receptor-9 597

signalling-accelerated glomerulonephritis. Clin Exp Immunol 166: 235-250, 2011 598

29. Schwaller J, Schneider P, Mhawech-Fauceglia P, McKee T, Myit S, Matthes T, 599

Tschopp J, Donze O, Le Gal FA, Huard B: Neutrophil-derived APRIL concentrated 600

in tumor lesions by proteoglycans correlates with human B-cell lymphoma 601

aggressiveness. Blood 109: 331-338, 2007 602

30. Burjanadze M, Matthes T, McKee T, Passweg J, Huard B: In situ detection of 603

APRIL-rich niches for plasma-cell survival and their contribution to B-cell 604

lymphoma development. Histol Histopathol 24: 1061-1066, 2009 605

31. Maia S, Pelletier M, Ding J, Hsu YM, Sallan SE, Rao SP, Nadler LM, Cardoso AA: 606

Aberrant expression of functional BAFF-system receptors by malignant B-cell 607

precursors impacts leukemia cell survival. PLoS One 6: e20787, 2011 608

32. Kern C, Cornuel JF, Billard C, Tang R, Rouillard D, Stenou V, Defrance T, 609

Ajchenbaum-Cymbalista F, Simonin PY, Feldblum S, Kolb JP: Involvement of 610

BAFF and APRIL in the resistance to apoptosis of B-CLL through an autocrine 611

pathway. Blood 103: 679-688, 2004 612

27

33. He B, Chadburn A, Jou E, Schattner EJ, Knowles DM, Cerutti A: Lymphoma B 613

cells evade apoptosis through the TNF family members BAFF/BLyS and APRIL. J 614

Immunol 172: 3268-3279, 2004 615

34. Gupta M, Dillon SR, Ziesmer SC, Feldman AL, Witzig TE, Ansell SM, Cerhan JR, 616

Novak AJ: A proliferation-inducing ligand mediates follicular lymphoma B-cell 617

proliferation and cyclin D1 expression through phosphatidylinositol 618

3-kinase-regulated mammalian target of rapamycin activation. Blood 113: 619

5206-5216, 2009 620

35. Chu VT, Enghard P, Schurer S, Steinhauser G, Rudolph B, Riemekasten G, Berek 621

C: Systemic activation of the immune system induces aberrant BAFF and APRIL 622

expression in B cells in patients with systemic lupus erythematosus. Arthritis 623

Rheum 60: 2083-2093, 2009 624

36. Chu VT, Enghard P, Riemekasten G, Berek C: In vitro and in vivo activation 625

induces BAFF and APRIL expression in B cells. J Immunol 179: 5947-5957, 2007 626

37. Sallustio F, Cox SN, Serino G, Curci C, Pesce F, De Palma G, Papagianni A, 627

Kirmizis D, Falchi M, Schena FP, European IgAN Consortium: Genome-wide scan 628

28

identifies a copy number variable region at 3p21.1 that influences the TLR9 629

expression levels in IgA nephropathy patients. Eur J Hum Genet 23: 940-948, 2015 630

38. Kim J, Gross JA, Dillon SR, Min JK, Elkon KB: Increased BCMA expression in 631

lupus marks activated B cells, and BCMA receptor engagement enhances the 632

response to TLR9 stimulation. Autoimmunity 44: 69-81, 2011 633

39. Katsenelson N, Kanswal S, Puig M, Mostowski H, Verthelyi D, Akkoyunlu M: 634

Synthetic CpG oligodeoxynucleotides augment BAFF- and APRIL-mediated 635

immunoglobulin secretion. Eur J Immunol 37: 1785-1795, 2007 636

40. Suzuki S, Nakatomi Y, Sato H, Tsukada H, Arakawa M: Haemophilus 637

parainfluenzae antigen and antibody in renal biopsy samples and serum of patients 638

with IgA nephropathy. Lancet 343: 12-16, 1994 639

41. Nagasawa Y, Iio K, Fukuda S, Date Y, Iwatani H, Yamamoto R, Horii A, Inohara H, 640

Imai E, Nakanishi T, Ohno H, Rakugi H, Isaka Y: Periodontal disease bacteria 641

specific to tonsil in IgA nephropathy patients predicts the remission by the 642

treatment. PLoS One 9: e81636, 2014 643

29

42. Suzuki Y, Suzuki H, Yasutake J, Tomino Y: Paradigm shift in activity assessment 644

of IgA nephropathy - optimizing the next generation of diagnostic and therapeutic 645

maneuvers via glycan targeting. Expert Opin Biol Ther 15: 583-593, 2015 646

43. Berthoux F, Suzuki H, Thibaudin L, Yanagawa H, Maillard N, Mariat C, Tomino Y, 647

Julian BA, Novak J: Autoantibodies targeting galactose-deficient IgA1 associate 648

with progression of IgA nephropathy. J Am Soc Nephrol 23: 1579-1587, 2012 649

44. Suzuki Y, Matsuzaki K, Suzuki H, Okazaki K, Yanagawa H, Ieiri N, Sato M, Sato T, 650

Taguma Y, Matsuoka J, Horikoshi S, Novak J, Hotta O, Tomino Y: Serum levels 651

of galactose-deficient immunoglobulin (ig) A1 and related immune complex are 652

associated with disease activity of IgA nephropathy. Clin Exp Nephrol 18: 770-777, 653

2014 654

45. Yasutake J, Suzuki Y, Suzuki H, Hiura N, Yanagawa H, Makita Y, Kaneko E, 655

Tomino Y: Novel lectin-independent approach to detect galactose-deficient IgA1 in 656

IgA nephropathy. Nephrol Dial Transplant 2015 657

46. Horie A, Hiki Y, Odani H, Yasuda Y, Takahashi M, Kato M, Iwase H, Kobayashi Y, 658

Nakashima I, Maeda K: IgA1 molecules produced by tonsillar lymphocytes are 659

under-O-glycosylated in IgA nephropathy. Am J Kidney Dis 42: 486-496, 2003 660

30

47. Inoue T, Sugiyama H, Hiki Y, Takiue K, Morinaga H, Kitagawa M, Maeshima Y, 661

Fukushima K, Nishizaki K, Akagi H, Narimatsu Y, Narimatsu H, Makino H: 662

Differential expression of glycogenes in tonsillar B lymphocytes in association 663

with proteinuria and renal dysfunction in IgA nephropathy. Clin Immunol 136: 664

447-455, 2010 665

48. Chen X, Liu H, Peng Y, He L, Zhang Y, Xie Y, Peng X, Liu C, Liu F: Expression 666

and correlation analysis of IL-4, IFN-gamma and FcalphaRI in tonsillar 667

mononuclear cells in patients with IgA nephropathy. Cell Immunol 289: 70-75, 668

2014 669

49. He L, Peng Y, Liu H, Yin W, Chen X, Peng X, Shao J, Liu Y, Liu F: Activation of 670

the interleukin-4/signal transducer and activator of transcription 6 signaling 671

pathway and homeodomain-interacting protein kinase 2 production by tonsillar 672

mononuclear cells in IgA nephropathy. Am J Nephrol 38: 321-332, 2013 673

50. He L, Peng Y, Liu H, Yin W, Chen X, Peng X, Shao J, Liu Y, Liu F: Th1/Th2 674

polarization in tonsillar lymphocyte form patients with IgA nephropathy. Ren Fail 675

36: 407-412, 2014 676

31

51. Suzuki H, Raska M, Yamada K, Moldoveanu Z, Julian BA, Wyatt RJ, Tomino Y, 677

Gharavi AG, Novak J: Cytokines alter IgA1 O-glycosylation by dysregulating 678

C1GalT1 and ST6GalNAc-II enzymes. J Biol Chem 289: 5330-5339, 2014 679

52. Brandtzaeg P, & Johansen FE: Mucosal B cells: Phenotypic characteristics, 680

transcriptional regulation, and homing properties. Immunol Rev 206: 32-63, 2005 681

53. Macpherson AJ, McCoy KD, Johansen FE, Brandtzaeg P: The immune geography 682

of IgA induction and function. Mucosal Immunol 1: 11-22, 2008 683

54. Suzuki H, Moldoveanu Z, Hall S, Brown R, Vu HL, Novak L, Julian BA, Tomana 684

M, Wyatt RJ, Edberg JC, Alarcon GS, Kimberly RP, Tomino Y, Mestecky J, 685

Novak J: IgA1-secreting cell lines from patients with IgA nephropathy produce 686

aberrantly glycosylated IgA1. J Clin Invest 118: 629-639, 2008 687

55. Imasawa T, Nagasawa R, Utsunomiya Y, Kawamura T, Zhong Y, Makita N, Muso 688

E, Miyawaki S, Maruyama N, Hosoya T, Sakai O, Ohno T: Bone marrow 689

transplantation attenuates murine IgA nephropathy: Role of a stem cell disorder. 690

Kidney Int 56: 1809-1817, 1999 691

32

56. Suzuki H, Suzuki Y, Aizawa M, Yamanaka T, Kihara M, Pang H, Horikoshi S, 692

Tomino Y: Th1 polarization in murine IgA nephropathy directed by bone 693

marrow-derived cells. Kidney Int 72: 319-327, 2007 694

57. van den Wall Bake AW, Daha MR, Radl J, Haaijman JJ, Van der Ark A, Valentijn 695

RM, Van Es LA: The bone marrow as production site of the IgA deposited in the 696

kidneys of patients with IgA nephropathy. Clin Exp Immunol 72: 321-325, 1988 697

58. van den Wall Bake AW, Daha MR, Evers-Schouten J, van Es LA: Serum IgA and 698

the production of IgA by peripheral blood and bone marrow lymphocytes in 699

patients with primary IgA nephropathy: Evidence for the bone marrow as the 700

source of mesangial IgA. Am J Kidney Dis 12: 410-414, 1988 701

59. Harper SJ, Allen AC, Pringle JH, Feehally J: Increased dimeric IgA producing B 702

cells in the bone marrow in IgA nephropathy determined by in situ hybridisation 703

for J chain mRNA. J Clin Pathol 49: 38-42, 1996 704

60. Moldoveanu Z, Wyatt RJ, Lee JY, Tomana M, Julian BA, Mestecky J, Huang WQ, 705

Anreddy SR, Hall S, Hastings MC, Lau KK, Cook WJ, Novak J: Patients with IgA 706

nephropathy have increased serum galactose-deficient IgA1 levels. Kidney Int 71: 707

1148-1154, 2007 708

33

61. Moore JS, Kulhavy R, Tomana M, Moldoveanu Z, Suzuki H, Brown R, Hall S, 709

Kilian M, Poulsen K, Mestecky J, Julian BA, Novak J: Reactivities of 710

N-acetylgalactosamine-specific lectins with human IgA1 proteins. Mol Immunol 711

44: 2598-2604, 2007 712

62. Matthes T, McKee T, Dunand-Sauthier I, Manfroi B, Park S, Passweg J, Huard B: 713

Myelopoiesis dysregulation associated to sustained APRIL production in multiple 714

myeloma-infiltrated bone marrow. Leukemia 29: 1901-1908, 2015 715

716

34

Table.1 Profiles of patients with IgA nephropathy (IgAN) and chronic 717

tonsillitis (CT) just before tonsillectomy 718

IgAN CTn 55 12age 35.1 31.4Male (％) 47.3 (26: 29) 50 (6: 6)Duration from onset to tonsillectomy(year)

8.5±8.8 ー

sCr (mg/dl) 0.86±0.3 0.6±0.14BUN (mg/dl) 13.1±3.2 11.5±3.6eGFR (ml/min/1.73m2) 79.5±28.1 116.4±26.7Proteinuria/urine Cr (g/gCr) 1.22±0.86 ーHematuria (RBC/HPF)

1-4/HPF 8 ー5-9/HPF 9 ー

10-15/HPF 3 ー16-20/HPF 11 ー21-25/HPF 5 ー26-30/HPF 3 ー

>30HPF 16 ー 719

Values are mean ±SD. 720

eGFR, estimated glomerular filtration rate. 721

Hematuria: Assessed by assigning scores according to number of 722

red blood cells per high-power field (RBC/HPF). 723

724

725

726

Table.2 The correlation between Stalk-1+ GC and the severity of the efficacy of 727

tonsillectomy in IgAN patients 728

Stalk -1+GC<10%(n=12)

Stalk -1+GC≧10%(n=43)

P Value

Proteinuria (g/gCr) before treatment 0.69±0.48 1.35±0.9 0.0298

Patients whose proteinuria decerasemore that 50％ after tonsillectomy (％)

25.0 (3/12) 62.8 (27/43) 0.0201 729

Figure 1A

APR

IL R

atio

/GAP

DH

IgAN CT0

2

4

6

8

10

★

Perc

enta

ge o

f GC

sta

ined

w

ith S

talk

-1

IgAN CT

D

0

20

40

60

80

100

★NS

CTIgAN

Stal

k-1＋

elas

tase

＋ce

ll/m

m2

C

B

IgAN

CT

Figure 2

A merge

Stalk-1

CD19

merge

Stalk-1

IgG

merge

Stalk-1

IgＭ

merge

Stalk-1

IgA

B

FSC

SSC CD19

Cel

l cou

nt

IgD

CD38 Stalk-1

Cel

l cou

nt

IgD

CD38 Stalk-1

Cel

l cou

nt

FSC

SSC CD19

Cel

l cou

nt

★★

CTIgAN0

5

10

15

20

Stal

k-1＋

GC

B c

ells

/GC

B c

ells

A

C

APRIL α

APRIL delta/zeta

H2OReh 1 2 3 4 1 2 3 4IgAN CT

APRIL alpha 1 mpasspfllapkgppgnmggpvrepalsvalwlswgaalgavacamalltqqtelqslrrAPRIL delta 1 ------------------------------------------------------------APRIL zeta 1 ------------------------------------------------------------

APRIL alpha 61 evsrlqgtggpsqngegypwqslpeqssdaleawengersrkrravltqkqkkqhsvlhlAPRIL delta 61 --------------------------•••••••••••••••••••••••••••-------APRIL zeta 61 -------------------------••••••••••••••••••••••••••••-------

APRIL alpha 121 vpinatskddsdvtevmwqpalrrgrglqaqgygvriqdagvyllysqvlfqdvtftmgqAPRIL delta 94 ------------------------------------------------------------APRIL zeta 93 ------------------------------------------------------------

APRIL alpha 181 vvsregqgrqetlfrcirsmpshpdraynscysagvfhlhqgdilsviipraraklnlspAPRIL delta 154 ------------------------------------------------------------APRIL zeta 153 ------------------------------------------------------------

APRIL alpha 241 hgtflgfvklAPRIL delta 214 ----------APRIL zeta 213 ----------

B merge

Stalk-1

Aprily-2

Figure 3

D

0

1

2

3★★

APR

ILα

Rat

io/G

APD

H

CTIgAN

Tonsillar B cells

0

1

2

3

4

APR

ILδ/

zeta

Rat

io/G

APD

H

CTIgAN

★★

★★

CTIgAN

TLR

9 R

atio

/GAP

DH

Figure 4

Tonsils Tonsillar B cells

APR

ILα

Rat

io/G

APD

H

TLR9 Ratio/GAPDH

B

R2=0.7611

P<0.0001

APR

IL δ

/zet

a R

atio

/GAP

DH

TLR9 Ratio/GAPDH

R2=0.7079

P<0.0001

★

TLR

9 R

atio

/GAP

DH

IgAN CT

A

Figure 5C

ellc

ount

Stalk-1 Aprily-2

A

5

5

Cel

lcou

nt

Stalk-1 Aprily-2

TACI

Cel

lcou

nt

BCMA

B

C

Aprily-2

Cel

lcou

nt

Stalk-1

≧10 <10Percentage of GC stained with Stalk-1

% d

ecre

ase

of s

erum

leve

ls o

f IgA

C P=0.052

≧10 <10

★★

Percentage of GC stained with Stalk-1

D

% d

ecre

ase

of s

erum

leve

ls o

f Gd-

IgA

Perc

enta

ge o

f GC

st

aine

d w

ith S

talk

-1

A

≧1g/gCr <1g/gCr

★

0

1

2

3

4

≧10 <10

★★

Percentage of GC stained with Stalk-1Pr

otei

nuria

(g/g

Cr)

B

Figure 6

IgA

UP

Gd-IgA1

% Stalk-1+GC

0

1

2

3

4

before tonsillectomy

after tonsillectomy

★

Prot

einu

ria (g

/gC

r)

E

0

1

2

3

4

before tonsillectomy

after tonsillectomy

★

Prot

einu

ria (g

/gC

r)

F

0.0

0.5

1.0

1.5

2.0

before tonsillectomy

after tonsillectomy

Prot

einu

ria (g

/gC

r)

GNS

Total GC≧10%

GC<10%

Figure 6

Self reproduction

B cells in tonsillar GC

exogenous antigensCpG

BCMA

TACI

TLR9endosomenuclei

sAPRIL(APRILα)

mAPRIL(APRILδ/zeta)

: Gd‐IgA1

Figure 7

Supplement Table.1

The correlation between Stalk-1+GC and the severity or the efficacy of tonsillectomy in severe

IgAN patients (proteinuria >0.5 g/gCr)

Supplement Table.2

Characteristics of IgAN patients before tonsillectomy and after tonsillectomy

ACEI, Angiotensin Coverting Enzyme InhibitorARB, Angiotensin Ⅱ Receptor Blocker

before tonsillectomy after tonsillectomy P Value

Systolic pressure (mmHg) 106.3±9.4 109.7±11.7 0.1553

Diastolic pressure (mmHg) 64.1±7.0 63.3±8.9 0.1983

Proteinuria/urine Cr (g/gCr) 1.22±0.86 0.60±0.64 <0.0001

Steroid therapy 1 1 1

The use of ACEIs and ARBs 19 21 0.6918

Stalk-1+GC<10%

(n=8)

Stalk-1+GC≧10%

(n=39)P Value

Proteinuria (g/gCr) before treatment 0.99±0.36 1.46±0.86 0.22

Patients whose proteinuria decrease

more that 50％ after tonsillectomy (％)28.6(1/8) 71.9 (26/39) 0.048