- Email: [email protected]
Characterizing Villitis of Unknown Etiology and Inflammation in Stillbirth
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62
The American Journal of Pathology, Vol. -, No. -, - 2016
ajp.amjpathol.org
Characterizing Villitis of Unknown Etiology and Inflammation in Stillbirth Q24
Hayley Derricott,* Rebecca L. Jones,* Susan L. Greenwood,* Gauri Batra,y Margaret J. Evans,z and Alexander E.P. Heazell* From the Maternal and Fetal Health Research Centre,* Institute of Human Development, University of Manchester, Manchester Academic Health Science Centre, Manchester; the Department of Paediatric Histopathology,y Royal Manchester Children’s Hospital, Manchester; and the Department of Paediatric Histopathology,z Edinburgh Royal Infirmary, Edinburgh, United Kingdom Accepted for publication December 8, 2015.
Q3
Q5
Address correspondence to Hayley Derricott, Maternal & Fetal Health Research Centre, 5th Floor St. Mary’s Hospital, Oxford Rd., Manchester M13 9WL, United Kingdom. E-mail: hayley.derricott@ manchester.ac.uk.
Villitis of unknown etiology (VUE) is an enigmatic inflammatory condition of the placenta associated with fetal growth restriction and stillbirth. Greater understanding of this condition is essential to understand how it contributes to adverse outcomes. Our aim was to identify and quantify the cells in VUE in cases of stillbirth and to characterize immune responses specific to this condition. Immunohistochemistry was performed on placentas from stillborn infants whose cause of death was recorded as VUE to identify CD45þ leukocytes, CD163þ macrophages, CD4þ T cells, CD8þ T cells, neutrophils, and proinflammatory and antiinflammatory cytokines. Images were quantified with HistoQuest software. CD45þ leukocytes comprised 25% of cells in VUE lesions: macrophages (12%) and CD4 T cells (11%) being the predominant cell types; CD8 T cells were also observed in all lesions. Leukocytes and macrophages were increased throughout the placenta in stillbirths; pan-placental CD4þ and CD8þ T cells outside VUE lesions increased in stillbirth with VUE. Increased IL-2 and IL-12 and reduced IL-4 immunostaining was seen in VUE lesions. Our results suggest VUE in stillbirth has a similar immune cell profile to live birth. Pan-placental macrophages, CD4 and CD8 T cells indicate a wider inflammatory response unrestricted to VUE lesions. The cytokine profile observed suggests a skew toward inappropriate Th1 immune responses. Full characterization of VUE lesion phenotype confirms its immunologic origins and provides foundations to develop novel investigations. (Am J Pathol 2016, -: 1e10; http://dx.doi.org/10.1016/j.ajpath.2015.12.010)
Stillbirth, the death of a baby after 20 weeks’ gestation, places a sustained economic and emotional burden on both maternity services and the wider community. Despite significant effort, the stillbirth rate has decreased at a slower rate than neonatal or infant death.1 In high-income countries stillbirth rates are variable. The United States and United Kingdom perform relatively poorly with rates of 1 in 160 and 1 in 212, respectively, whereas in Finland 1 in 400 pregnancies result in a stillborn infant.2e4 Placental dysfunction as a cause of stillbirth has increasingly become a focus of research effort with recognition that placental disorders are the cause of death in up to 65% of stillbirths.5 However, the placental lesions associated with stillbirth are extremely varied and poorly defined, and their relation to pathologic processes leading to stillbirth is difficult to assess.6,7 Placental lesions associated with stillbirth include those with genetic, environmental, infective, inflammatory, mechanical, metabolic, and vascular origins.6
Understanding of inflammatory disorders of the placenta remains relatively limited in comparison with vascular lesions. Two main inflammatory conditions have been linked to placental dysfunction in the absence of infection: villitis of unknown etiology (VUE) and chronic histiocytic intervillositis.8 VUE is an inflammatory condition of the placenta reported to occur in up to 15% of term placentas and more frequently in pregnancies resulting in poor outcome.9,10 The origin of VUE is unclear, although it is proposed to be maternal immune rejection of a semiallogeneic placenta.11e13 VUE is identified and characterized by the presence of elevated numbers of fetal macrophages (Hofbauer cells) and an infiltrate of maternal T lymphocytes in the villous stroma. Areas of the placenta not affected by VUE lesions display apparently normal structural characteristics.9 The cell profile of VUE has Supported by Tommy’sethe Baby Charity (H.D.). Disclosures: This work forms part of a Ph.D. project for H.D.
Copyright ª 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2015.12.010
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 Q4 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 Q1 Q2 123 124
125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186
Derricott et al Table 1
Antibodies and Positive Control Tissues Used for Identifying Immune Cell Populations and Cytokines in Placental Villous Tissue
Antigen
Antigen retrieval
Supplier
Working concentration (mg/mL)
Ab type
Control tissue
CD45 CD163 CD4 (clone 4B12) CD8 NE IL-2 IL-4 IL-6 IL-10 IL-12 TGF-b
Sodium citrate Sodium citrate Tris-EDTA or sodium citrate Tris-EDTA None Sodium citrate Sodium citrate Sodium citrate None Sodium citrate Sodium citrate
Dako AbD Serotec Dako Dako Dako Novus Biologicals Thermo Scientific Novus Biologicals Abcam Sigma-Aldrich Novus Biologicals
0.5 10 4.6 1.6 1.1 4.5 2.5 9.6 5 2.5 10
M mAb M mAb M mAb M mAb M mAb R pAb R pAb M mAb M mAb R pAb M mAb
Tonsil Tonsil Tonsil Tonsil PTLI Tonsil Tonsil Tonsil Liver (Kupffer cells) Trophoblast Tonsil
M mAb, mouse monoclonal antibody; NE, neutrophil elastase; PTLI, preterm labor with infection; R pAb, rabbit polyclonal antibody; TGF, transforming growth factor.
previously been characterized in both appropriately grown and growth-restricted live born infants.12,14e16 Conflicting reports state either CD4þ or CD8þ T cells are the most common lymphocyte type in VUE lesions.12,14,17,18 The subtype of CD4þ cells (Th1/Th2) has not been examined; therefore, the type of immune response in VUE remains to be elucidated. A thorough characterization of VUE has yet to be completed in stillbirth; it is not understood why it may cause fetal demise. In addition, the cytokine profile in VUE has not been described in either live births or stillbirths. Characterization of the cell types present and their cytokine profile will provide a greater understanding of the pathophysiology of VUE. We hypothesized that the immune cells in VUE lesions in stillbirth would be comparable with those described in live birth and that evidence of widespread inflammatory changes would be detectable in the placentas of stillborn infants with VUE. In this immunomorphologic study we aimed to accurately quantify the type and number of immune cells present in VUE lesions, assess leukocyte infiltration across the placenta, and analyze the expression of a range of proinflammatory and anti-inflammatory cytokines with the use of immunohistochemical approaches, coupled with an unbiased sensitive image analysis method. We compared leukocyte infiltration and cytokine expression across the placenta to ascertain whether this was altered in Table 2
dysfunctional placentas from stillbirth with and without VUE and fetal growth restriction (FGR) compared with healthy controls.
Materials and Methods Study Population Placental tissues from pregnancies resulting in stillbirth in which a contributory cause of death was recorded as VUE (n Z 12) or FGR (n Z 12) were obtained from the Pediatric Histopathology departments of Manchester Royal Children’s Hospital and Edinburgh Royal Infirmary. Samples from stillborn growth-restricted infants had no evidence of VUE lesions recorded on postmortem examination. Consent for tissue use in research was obtained at the time of consent for postmortem; their use in this project was approved by Humber Bridge Research Ethics Committee (Ref. 13/YH/0176). Live born infants (n Z 11) with an individualized birth weight centile less than the fifth were considered FGR infants; placental tissue was obtained from the Maternal and Fetal Health Research Group Biobank, University of Manchester (North West REC 08/H1010/55). Matched placental samples from women with uncomplicated pregnancies (n Z 12) were selected from the Maternal and Fetal Health Research
Demographic Information for the Women Included in the Study
Characteristic
Stillbirth with VUE (n Z 12)
Stillbirth with FGR (n Z 12)
Live birth with FGR (n Z 11)
Normal pregnancy (n Z 12)
P
Maternal age (years) Gestational age (weeks) Gravidity Parity Birthweight (g) IBC
32 (21e36) 39 (20e41) 3 (1e7) (n Z 7) 1 (0e3) (n Z 7) NA 23 (3e90) (n Z 7)
23.5 35.21 1 0 1061 NA
32 37.8 1 0 2246 3
35 33.5 2 1 3430 45
0.019 0.098 0.298 0.118 <0.001 <0.001
(19e42)* (28e38) (1e4) (0e2) (385e2000)***
(16e35) (27.7e42.1) (1e9) (0e3) (747e3060)** (0e10)***
(25e42) (30.0e41.7) (1e18) (0e3) (2900e3840) (12e93)
Results are presented as median (range). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with normal pregnancy. FGR, fetal growth restriction; IBC, individualized birth weight centile; NA, not available; VUE, Villitis of unknown etiology.
2
ajp.amjpathol.org
-
The American Journal of Pathology
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
Q23
187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248
extensive CD45þ cell infiltrate that displayed the morphologic properties of macrophages and lymphocytes. Subsequent immunostaining for specific immune cells was performed to identify CD4þ T cells, CD8þ T cells, CD163þ macrophages, and neutrophils. Further immunostaining for cytokines IL-2, IL-4, IL-10, and IL-12 was performed on representative sections from all samples. Finally, staining for IL-6 and transforming growth factor (TGF)-b was performed on selected stillbirth samples with VUE (n Z 6) and healthy pregnancies (n Z 6). Heat-mediated antigen retrieval was performed for all antibodies except antieneutrophil elastase and antieIL-10 monoclonal antibodies. Primary antibodies were applied at the working concentrations detailed in Table 1. ½T1 Each tissue section had a corresponding negative control on which mouse or rabbit IgG (Sigma-Aldrich) was applied at the Q8 same concentration as the primary antibody. Each staining run included a positive control tissue as described in Table 1.
print & web 4C=FPO
249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310
Characterizing VUE in Stillbirth
Q15
Q16
Immunofluorescence
Figure 1 Serial images of a representative VUE lesion immunostained for immune cell markers. CD45 (A), CD163þ macrophages (B), CD8 cytotoxic T cells (C), CD4þ T helper cells (D), neutrophil elastase (E), and negative (F). Arrows highlight examples of positively stained cells. Scale bar Z 50 mm. Original magnification, 100. VUE, villitis of unknown etiology.
Group Biobank and used as controls. All placentas were taken from mothers who showed no evidence of conditions such as preeclampsia or diabetes. Detailed case note review showed no clinical, biochemical, hematologic, or microbiological evidence of infection was present. Multiple pregnancies, abnormal fetal heartbeat (live birth only), and fetuses with anomalies were excluded. Q6
Sample Collection Placentas from cases of stillbirth had two to seven randomly selected samples per placenta obtained at the time of postmortem for histologic analysis. For live births three samples of placental villous tissue were taken, one each from the center, middle, and periphery of the tissue.19 Biopsies were fixed in neutral-buffered formalin for 24 hours before being processed and embedded in paraffin wax.
IHC
Q7
For immunohistochemistry (IHC), 5-mm serial sections were cut and mounted onto slides coated with poly-L-lysine (Sigma-Aldrich, Poole, UK). Immunostaining with colorimetric detection was performed as described previously.20 VUE lesions were initially identified by detecting clusters of leukocytes with the use of a mouse monoclonal antibody to CD45 (leukocyte common antigen). A VUE lesion was defined as an area of villous tissue with evidence of an
The American Journal of Pathology
-
The phenotype of the macrophages and T cells in the placentas/ lesions was investigated by dual immunofluorescence with combinations of immune cell markers CD4 and CD163 and cytokines IL-2, IL-12, or IL-4. Sections were fixed and processed as described in the previous section. Autofluorescence was quenched by 1-hour incubation in 0.25% ammonia (SigmaAldrich) in 70% ethanol. Primary antibodies were applied at the concentrations described in Table 1 at 4 C overnight. Secondary antibodies (goat anti-mouse Alexa 568 or goat antirabbit Alexa 488; Life Technologies, Paisley, UK) were applied at 5 mg/mL and incubated for 30 minutes at room temperature. DAPI-containing aqueous mounting medium was used to apply coverslips.
Image Capture and Analysis Tissue sections were visualized with the 10 objective of an Olympus BX41 light microscope (Southend-on-Sea, UK). Images were captured with QIcam Fast 1394 camera (QImaging, Surrey, BC, Canada) and Image Pro Plus 6.0 (Media Cybernetics Inc., Rockville, MD). Quantitative, unbiased image analysis was completed with HistoQuest image analysis software (TissueGnostics, Vienna, Austria). The Q9 pixels of images viewed in this software are converted to grayscale (0 to 256 scale) and assigned an arbitrary number relating to staining intensity. The image analysis software differentiates hematoxylinþ and diaminobenzidine (DAB)þ Q10 cells or areas that are recorded as events (cells/nuclei) or area of staining. Results are presented as histograms or dot plot scattergrams to which staining area and/or intensity cutoffs can be applied to differentiate between cell populations. Analysis variables were optimized such that the number of DABþ events and hematoxylinþ events were counted. Two analyses were performed. First, for characterization of VUE lesions in stillbirth, inflammatory foci were identified in CD45-stained sections, then the remaining immunostains were
ajp.amjpathol.org
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
3
311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372
Figure 2 AeC: Screenshots from HistoQuest image analysis software demonstrating the selection of DABþ events in sections stained for CD45 leukocyte common antigen (A and B) and cytotoxic T-lymphocyte marker CD8 (C). Positive events are highlighted in red; events encircled in green indicate hematoxylin positive/DAB events. D: A representative scattergram from which staining intensity variables were ascertained. Scale bar Z 50 mm. Original magnification, 100. DAB, diaminobenzidine.
print & web 4C=FPO
373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434
Derricott et al
Q11
Q17 Q18
Q19
matched and captured in serial sections. In tissue sections with widespread/numerous inflammatory foci, representative lesions were selected (n Z 2 to 5 per section). To assess pan-placental inflammation in tissue, 10 random images (excluding lesions) were taken for each placental slide from all groups (stillbirth with VUE, stillbirth with FGR, live birth with FGR, and controls), immunostained for CD45, CD163 macrophage, CD4 T cell, and CD8 T cell markers. DABþ cells and nuclei were quantified to ascertain whether there were any differences in leukocyte numbers between the study groups. The presence of cytokines was determined by immunostaining for IL-2, IL-4, IL-6, IL-10, IL-12, and TGF-b. Images were analyzed quantitatively in sections of tissue from VUE lesions, areas of stillbirth with VUE placentas that were devoid of lesions, stillbirth with FGR placentas, live birth with FGR placentas, and controls (n Z 10 images per placenta). Area and intensity of staining were recorded for each placental sample, and quantitative analysis was completed with HistoQuest image analysis software. Fluorescent-stained sections were visualized with a Zeiss fluorescence microscope, AxioCam MRn camera with Zen pro image processing software (Zeiss, Welwyn Garden City, UK).
(GraphPad Software, San Diego, CA). Nonparametric categorical data were analyzed with Fisher’s exact test; nonparametric continuous data were analyzed with U test or Kruskal-Wallis test with Dunn’s post hoc test.
Statistical Analysis
Characterization of Focal Inflammatory Lesions
We powered our analysis to determine the number of CD45þ and CD163þ cells in placentas from stillborn infants, assuming similar increases in these cells as seen in placentas from poor outcome pregnancies experiencing reduced fetal movements.21 No similar studies have been conducted with CD4 and CD8 T cells, so we were not able to take direct comparisons. For 80% power with an a value of 0.05, the sample size was calculated as 12. All statistical analysis was performed with GraphPad Prism software version 6.0
VUE lesions were identified in all placentas from the stillbirth with VUE cohort; a representative lesion is shown in Figure 1. In total 104 VUE lesions from 12 placentas were ½F1 photographed and analyzed. The number of CD45þ cells in the lesions contributed 25% (median; range, 8% to 48%) to the total number of nuclei. Macrophages and CD4þ T lymphocytes were the most prevalent immune cells in the lesions, accounting for 12% (9% to 23%) and 11% (3% to 14%) of the total number of nuclei, respectively. CD8þ T
4
Results Participant Demographic Characteristics The demographic data for the 48 women included in the study are shown in Table 2. A significant difference was found in ½T2 maternal age between the stillbirth with FGR and normal pregnancy groups (P Z 0.02). Contributory causes of death for the stillborn infant cohorts are detailed in Supplemental Table S1. By experimental design birth weight and individualized birth weight centile differences between stillbirth with FGR, live birth with FGR, and normal pregnancies were highly significant (P < 0.001 and P < 0.01, respectively). Average time between fetal death and delivery of infant was 3.5 days (range, 1 to 14 days). Comparisons of the grading/ extent of VUE between tissue samples could not be drawn because this information was not recorded in postmortem reports (Supplemental Table S2).
ajp.amjpathol.org
-
The American Journal of Pathology
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496
lymphocytes were also present in VUE lesions, contributing 4% (0.3% to 11%) to the total number of nuclei. Neutrophil levels were low in all samples, accounting for 1% (0.37% to 2%) of total number of nuclei.
Differentiating Cell Populations Cell populations were differentiated by applying staining area and/or intensity cutoffs to the results presented as his½F2 tograms or dot plot scattergrams (Figure 2).
print & web 4C=FPO
497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558
Characterizing VUE in Stillbirth
The American Journal of Pathology
-
559 560 561 562 563 564 565 566 Figure 3 Quantification of pan-placental im567 mune cells in normal and pathologic pregnancies. 568 CD45 (A), macrophages (CD163, B), cytotoxic 569 T cells (CD8, C), and T helper cells (CD4; D). 570 Horizontal lines represent median. *P < 0.05, **P < 0.01, and ***P < 0.001, Kruskal-Wallis test 571 with Dunn’s post hoc test. LB FGR, live birth with 572 fetal growth restriction; NP, normal pregnancy; SB 573 FGR, stillbirth with fetal growth restriction; SB 574 VUE, stillbirth with villitis of unknown etiology. 575 576 577 578 579 580 581 582 583 584 Quantification of Pan-Placental Immune Cells 585 586 Significant increases were detected in the number of pan587 placental CD45þ leukocytes in all placentas from pregnancies 588 from stillborn infants (with or without VUE) compared with 589 normal pregnancies (P < 0.001) (Figure 3A). Significant ½F3 590 elevation in macrophage numbers was detected in the pla591 centas from stillborn infants with VUE (P < 0.01), stillborn 592 593 infants with FGR (P < 0.001), and live born infants with FGR 594 (P < 0.01) compared with normal pregnancy (Figure 3B). 595 Placentas from the stillbirth with VUE group showed 596 597 598 599 600 601 602 Figure 4 Representative images of placental 603 sections immunostained for proinflammatory and 604 anti-inflammatory cytokines. A, D, and G: VUE lesions were stained for IL-2 (A), IL-12 (D), and IL-4 605 (G). B, E, and H: Cytokine staining in areas of 606 placenta unaffected by lesions from stillborn in607 fants with VUE were stained for IL-2 (B), IL-12 (E), 608 and IL-4 (H). C, F, and I: Representative sections 609 of placenta from pregnancies with a normal 610 outcome were stained for IL-2 (C), IL-12 (F), and 611 Q20 IL-4 (insets, negatives) (I). Arrows highlight 612 staining associated with cells; arrowheads high613 light staining associated with trophoblast. Scale 614 bar Z 50 mm. Original magnification, 100. VUE, villitis of unknown etiology. 615 616 617 618 619 620
ajp.amjpathol.org
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
5
Derricott et al
print & web 4C=FPO
621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 Figure 5 Representative high power images of placental sections after 646 immunostaining for proinflammatory and anti-inflammatory cytokines. 647 A, C, and E: VUE lesions were stained for IL-2 (A), IL-12 (C), and IL-4 (E). B, D, and F: Representative sections of placenta from pregnancies with a 648 normal outcome were stained for IL-2 (B), IL-12 (D), and IL-4 (F). Arrows 649 highlight staining of cells in the stromal core, arrowheads highlight 650 staining associated with syncytiotrophoblast. Scale bar Z 20 mm. Original 651 magnification, 400. VUE, villitis of unknown etiology. 652 653 654 significantly more pan-placental CD4 and CD8 cells than all 655 other groups (P < 0.001) and were observed in every field of 656 view, excluding lesions (Figure 3, C and D). 657 658 659 Cytokines 660 661 To fully characterize the immune response in VUE, im662 munostaining of characteristic Th1 (IL-2, IL-12) and Th2 663 (IL-4) cytokines was performed (Figure 4). Staining of IL-2 ½F4 664 and IL-12 localized to cells within the stromal core and 665 stillborn, as previously shown22 (Figure 5). Area of IL-12 ½F5 666 667 staining was significantly increased in VUE lesions and 668 live born FGR placentas compared with normal pregnancy 669 (P < 0.001). A significant increase in area of IL-2 staining 670 ½F6 was observed in VUE lesions (P < 0.05) (Figure 6). IL-4 671 staining localized to both stromal and trophoblast cells23 672 (Figure 5). A significant reduction was found in the area 673 of IL-4 immunostaining in VUE lesions and areas devoid of 674 lesions compared with controls (P < 0.05 and P < 0.01, 675 respectively). A significant reduction in IL-4 staining area 676 was also observed in the stillbirth with FGR group 677 compared with live birth with FGR and controls (P < 0.05 678 679 and P < 0.001, respectively). IL-10 appeared significantly 680 increased overall in areas of VUE placentas devoid of 681 lesions (P < 0.001), but individual comparisons failed to 682
6
reach statistical significance (Figures 4 and 6, Supplemental Figure S1). Staining intensity for all cytokines was significantly different between samples (Supplemental Figure S2). Intensity of IL-2 was significantly reduced (stillbirth FGR, P Z 0.006; live birth FGR, P Z 0.0004) and IL-12 increased (stillbirth FGR, P Z 0.0002; live birth FGR, P < 0.001) in FGR samples compared with control. IL-4 intensity was significantly reduced in VUE lesions compared with control (P Z 0.02). IL-10 staining intensity was significantly increased in VUE placentas compared with stillbirth with FGR (VUE lesion, P Z 0.03; VUE nonlesion, P Z 0.01). Immunostaining for cytokines IL-6 and TGF-b was not detected in VUE lesions (Supplemental Figure S1).
Macrophage and T-Cell Phenotype Dual immunofluorescence was performed to identify immune cell sources of cytokines of interest. IL-2 co-localized with CD4þ staining, CD163þ on the cell surface co-localized with cytoplasmic IL-12 staining, and IL-4 co-localized with CD4þ (Figure 7). Evidence of faint IL-4 fluorescence was found in ½F7 the syncytiotrophoblast layer as observed in the images of chromogen staining (Figures 4 and 7).
Discussion This study has fully characterized VUE in placentas from stillborn infants by quantifying the immune cells found in the lesions by using unbiased image analysis software, after IHC to detect specific immune cell types. We have also demonstrated specific pan-placental inflammatory changes in stillbirths (with and without VUE), indicating a more widespread immune reaction occurring with localized VUE lesions. Furthermore, our cytokine analyses indicate that the immune response in the lesions is directed toward Th1-type response. Previous studies of VUE in live birth have described lesions comprising mainly fetal macrophages24 and maternal CD4þ18,25 or CD8þ12,14 T lymphocytes. Our results support these descriptions: in cases of stillbirth, macrophages and CD4þ T cells predominate, although elevated numbers of CD8 cells are also present. A key strength of the current study is the use of immunostaining to detect specific immune cell types. Pathologists examining hematoxylin and eosin-stained sections would not be able to identify individual leukocyte subsets, which are clearly visible after immunostaining for leukocyte, macrophage, CD4, and CD8 cell markers. VUE has been likened to graft rejection or graft-versus-host disease.11e13 The phenotype that we observed, with CD4þ lymphocytes outnumbering CD8þ lymphocytes, agrees with the 1989 study of Labarrere et al25 and supports this hypothesis because the T-helper cells would assume responsibility for directing the immune response toward a Th1-type response.26,27 The presence of cytotoxic CD8 T cells in the lesions may contribute to both
ajp.amjpathol.org
-
The American Journal of Pathology
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744
745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806
Characterizing VUE in Stillbirth
Figure 6 Quantification of cytokine staining area using HistoQuest image analysis software. AeD: IL-2 (A), IL-12 (B), IL-4 (C), and IL-10 (D). Horizontal lines represent median. *P < 0.05, **P < 0.01, and ***P < 0.001, Kruskal-Wallis test with Dunn’s post hoc test. DAB, diaminobenzidine; LB FGR, live birth with fetal growth restriction; NP, normal pregnancy; SB FGR, stillbirth with fetal growth restriction; VUE, villitis of unknown etiology.
the fetal placental vasculopathy often observed in VUE and the recently described elevated apoptosis of intravillous cells.9,17,28,29 Since Medawar30 first described the immunologic paradox of pregnancy, much has been published on the shift from Th1 to Th2 responses in pregnancy.31e35 Suppression of antiefetal cell-mediated responses and a skew toward antibody-mediated immunity appears to be advantageous in the maintenance of a successful pregnancy.35,36 The cytokine profile differs between Th1 and Th2 responses; IL-2, IL-6, and IL-12 are associated with Th1 responses, whereas anti-inflammatory cytokines IL-4, IL-10, and TGF-b predominate in Th2-type responses. Our findings of increased levels of IL-2 and IL-12 in VUE lesions suggest that there may be an inappropriate shift toward cell-mediated immunity in VUE cases associated with stillbirth. Staining intensity of IL-2 and IL-12 was not significantly different between VUE samples and controls, implying that more cells are secreting the cytokine but the expression of it is not different. Co-localized immunofluorenscence staining of CD163/IL-12 and CD4/IL-2 is indicative of proinflammatory macrophage and Th1 CD4 T-cell phenotypes. We have also detected a significant reduction in levels of anti-inflammatory IL-4 in VUE placentas from cases of stillbirth. The reduction in IL-4 levels was mirrored by a significant reduction in expression in VUE lesions. The placental anti-inflammatory cytokine environment characterized by IL-4, IL-10, and TGF-b, which induces maternal tolerance of the fetus, is
The American Journal of Pathology
-
critical in maintaining a healthy pregnancy.32,36,37 Conversely, levels of proinflammatory IL-12 remain low throughout successful pregnancy.36 Significant decreases in IL-4 in stillbirth placentas may be a contributory factor in permitting a shift from Th2-type to Th1-type immunity. These data support a reduction in Th2 and a Th1 bias in VUE lesions, which may lead to unresolved cell-mediated inflammation. Therefore, the critical balance appears to be disrupted in VUE lesions in cases ending in stillbirth. It has been stated that macrophages and their cytokines are fundamental in establishing and maintaining the placenta at the start of pregnancy.38,39 It is therefore reasonable to suggest that an excess of immune cells and proinflammatory cytokines in late pregnancy may have a detrimental effect on placental function, which is then manifested as FGR or stillbirth. These effects would be in addition to the cytotoxic effects of any immune response mounted. Interestingly, systematic reviews suggest a general shift toward a proinflammatory cytokine profile in maternal serum and placentas from FGR and small for gestational age infants, confirming a role for a proinflammatory state in FGR.40,41 Conflicting reports of M2 placental macrophages potentially acting as either proinflammatory42 or immunosuppressive29 cells only serve to compound the mystery underlying VUE. Our investigation has shown placental macrophages expressing the M2 marker CD163, yet producing the proinflammatory cytokine IL-12, further enhancing the evidence of the
ajp.amjpathol.org
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
7
Q21
807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868
Figure 7
Representative high power images of placental sections after dual immunofluorescence for proinflammatory and anti-inflammatory cytokines and cell markers. AeC: VUE lesions stained for CD4 (red), IL-2 (green), co-localization (yellow) (A), CD163 (red) and IL-12 (green) (B), and CD4 (red) and IL-4 (green), co-localization (yellow) (C). D: Mouse and rabbit IgG isotype control. Red arrows highlight dual staining; yellow arrows indicate co-localized immunofluorescence; and white arrowheads indicate trophoblast-localized IL-4. Scale bar Z 20 mm. Original magnification, 630. VUE, Villitis of unknown etiology.
print & web 4C=FPO
869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930
Derricott et al
Q12
plasticity of these cells and their potential role in pregnancy complications. Pan-placental increases in leukocyte numbers in VUE are a novel finding. Significantly elevated numbers of leukocytes and macrophages were detected in all stillbirths compared with controls. The elevation in macrophages in live born FGR suggests that this is not a postmortem artifact. These data are consistent with our recent findings of elevated placental macrophage numbers in pregnancies at high risk of stillbirth.21 Staining intensity of cytokines in both live born and stillborn FGR were consistent throughout, further confirming that the structural characteristics observed is not related to postmortem tissue changes. The placentas from these pregnancies also exhibited a proinflammatory bias in relation to cytokine mRNA and protein expression (elevated IL-1 family members, reduced IL-10 and IL-4). Together, these data are highly suggestive of placental inflammation being an underlying factor in pregnancies that result in FGR and stillbirth. Previous descriptions of VUE have stated the occurrence of focal villous lesions with adjacent unaffected tissue displaying normal structural characteristics.9 It has been suggested that the occurrence of VUE may have been underestimated because sampling of the placenta was not extensive enough to find evidence of lesions43; Altemani et al43 reported that at least six blocks are needed to achieve an 85% chance of diagnosing 95% of cases. In our study, CD4 and CD8 T-cell numbers were increased in stillbirths with VUE compared with all other group samples, and these increases were not confined to VUE lesions. This finding is a strength of the specificity of the IHC in our study. A sensitive and unbiased image analysis tool that can accurately detect immunostained cells with the use of a relatively low magnification (100) also contributes to the strength of the study. Our data suggest that a pan-placental increase in
8
CD4 and CD8 T cells should stimulate the pathologist to suspect an inflammatory condition, such as VUE, without relying on locating a specific lesion. With the use of IHC and sensitive image analysis software such as HistoQuest, the incidence of VUE could potentially be identified and accurately reported with fewer tissue samples. A general consensus on the sampling and definitions of placental lesions, as discussed by the Amsterdam Placental Workshop Group, attempts to address issues such as this (T.V. Khong et al, unpublished data). More sophisticated placental pathologic investigations may improve diagnosis of VUE (as was the case with estrogen receptor expression in breast cancer44) and thus provide much sought after explanations for the cause of death. By examining placental tissue after delivery, IHC can only provide a snapshot of VUE, rather than a temporal profile of the cell migration and its associated effects. This could explain why descriptions of the proportions of T cells in VUE differ between research studies because they sample different periods in the evolution of lesions.12,14,17,18 In addition, the panplacental presence of CD4 and CD8 T cells may represent pre-lesions that, given time, would expand within the villous stroma, potentially explaining why VUE is considered a thirdtrimester phenomenon.9 The 1995 study by Khong,45 describing expression of major histocompatibility complex type II in both nonvillitis and villitis-affected placentas, also suggests that VUE may be an evolving lesion. This dynamic process cannot be surmised from the current method of studying placental tissue. The results of this study would be further enhanced by including samples of placenta from live born infants with evidence of VUE. At the time the study was conducted, these were not available. A greater understanding of the immune responses associated with VUE may lead to the development of screening
ajp.amjpathol.org
-
The American Journal of Pathology
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
Q22
931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992
993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054
Characterizing VUE in Stillbirth tests to enable antenatal detection of VUE. Our laboratory recently published data that indicate alterations in concentrations of maternal serum cytokines in pregnancies characterized by placental inflammation.21 This is supported by the observation of elevated maternal plasma chemokines (CXCL9, CXCL10, and CXCL11) in cases of VUE.13 It may therefore be possible to identify placental inflammation antenatally which would allow increased antenatal surveillance and potential anti-inflammatory therapies to prevent stillbirth.
7.
8. 9. 10.
11.
Conclusion We have fully characterized VUE in stillbirths with the use of IHC to identify specific immune cell types and have detected evidence of a Th1-type cytokine response. These data present novel insights into the pathogenesis of VUE and provide solid foundations for functional studies to determine the impact of inflammation on placental function.
12.
13.
Acknowledgments 14.
We thank the mothers who gave consent to use the placentas that we studied, the recruiters, Dr. Mark Wareing who coordinated the Maternal and Fetal Health Research Group Biobank, Dr. Sylvie Girard for allowing us to use some of the immunostained sections from healthy pregnancies, Dr. Rachael Wright who dedicated a lot of time optimizing antibodies for IHC, and Dr. Joanne Pennock for immunologic advice.
15.
16.
17.
Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2015.12.010.
18.
19.
References 1. Flenady V, Middleton P, Smith GC, Duke W, Erwich JJ, Khong TY, Neilson J, Ezzati M, Koopmans L, Ellwood D, Fretts R, Frøen JF; Lancet’s Stillbirths Series steering committee: Stillbirths: the way forward in high-income countries. Lancet 2011, 377:1703e1717 2. MacDorman M, Kirmeyer S: Fetal and perinatal mortality, United States, 2005. Natl Vital Stat Rep 2009, 57:1e19 3. Lawn JE, Blencowe H, Pattinson R, Cousens S, Kumar R, Ibiebele I, Gardosi J, Day LT, Stanton C; Lancet’s Stilbirths Series steering committee: Stillbirths: Where? When? Why? How to make the data count? Lancet 2011, 377:1448e1463 4. Office of National Statistics: Statistical Bulletin: Births in England and Wales, 2013. Titchfield, United Kingdom, Office of National Statistics, 2014 5. Flenady V, Frøen JF, Pinar H, Torabi R, Saastad E, Guyon G, Russell L, Charles A, Harrison C, Chauke L, Pattinson R, Koshy R, Bahrin S, Gardener G, Day K, Petersson K, Gordon A, Gilshenan K: An evaluation of classification systems for stillbirth. BMC Pregnancy Childbirth 2009, 9:24 6. Heazell AEP, Worton SA, Higgins LE, Ingram E, Johnstone ED, Jones RL, Sibley CP: IFPA Gábor Than Award Lecture: recognition of
The American Journal of Pathology
-
20.
21.
22.
23.
24.
placental failure is key to saving babies’ lives. Placenta 2015, 36(Suppl 1):S20eS28 Ptacek I, Sebire NJ, Man JA, Brownbill P, Heazell AEP: Systematic review of placental pathology reported in association with stillbirth. Placenta 2014, 35:552e562 Redline RW: Placental inflammation. Semin Neonatol 2004, 9: 265e274 Redline RW: Villitis of unknown etiology: noninfectious chronic villitis in the placenta. Hum Pathol 2007, 38:1439e1446 Derricott H, Jones RL, Heazell AEP: Investigating the association of villitis of unknown etiology with stillbirth and fetal growth restriction a systematic review. Placenta 2013, 34:856e862 Rudzinski E, Gilroy M, Newbill C, Morgan T: Positive C4d immunostaining of placental villous syncytiotrophoblasts supports hostversus-graft rejection in villitis of unknown etiology. Pediatr Dev Pathol 2013, 16:7e13 Kim J-S, Romero R, Kim MR, Kim YM, Friel L, Espinoza J, Kim CJ: Involvement of Hofbauer cells and maternal T cells in villitis of unknown aetiology. Histopathology 2008, 52:457e464 Kim MJ, Romero R, Kim CJ, Tarca AL, Chhauy S, LaJeunesse C, Lee D-C, Draghici S, Gotsch F, Kusanovic JP, Hassan SS, Kim J-S: Villitis of unknown etiology is associated with a distinct pattern of chemokine up-regulation in the feto-maternal and placental compartments: implications for conjoint maternal allograft rejection and maternal anti-fetal graft-versus-host disease. J Immunol 2009, 182: 3919e3927 Brito H, Juliano P, Altemani C, Altemani A: Is the immunohistochemical study of the inflammatory infiltrate helpful in distinguishing villitis of unknown etiology from non-specific infection villitis? Placenta 2005, 26:839e841 Redline RW, Patterson P: Villitis of unknown etiology is associated with major infiltration of fetal tissue by maternal inflammatory cells. Am J Pathol 1993, 143:473e479 Kapur P, Rakheja D, Gomez AM, Sheffield J, Sanchez P, Rogers BB: Characterization of inflammation in syphilitic villitis and in villitis of unknown etiology. Pediatr Dev Pathol 2004, 7:453e458. discussion 421 Boog G: Chronic villitis of unknown etiology. Eur J Obstet Gynecol Reprod Biol 2008, 136:9e15 Katzman PJ, Murphy SP, Oble DA: Immunohistochemical analysis reveals an influx of regulatory T cells and focal trophoblastic STAT-1 phosphorylation in chronic villitis of unknown etiology. Pediatr Dev Pathol 2011, 14:284e293 Warrander LK, Batra G, Bernatavicius G, Greenwood SL, Dutton P, Jones RL, Sibley CP, Heazell AEP: Maternal perception of reduced fetal movements is associated with altered placental structure and function. PLoS One 2012, 7:e34851 Hamilton S, Oomomian Y, Stephen G, Shynlova O, Tower CL, Garrod A, Lye SJ, Jones RL: Macrophages infiltrate the human and rat decidua during term and preterm labor: evidence that decidual inflammation precedes labor. Biol Reprod 2012, 86:39 Girard S, Heazell AE, Derricott H, Allan SM, Sibley CP, Abrahams VM, Jones RL: Circulating cytokines and alarmins associated with placental inflammation in high-risk pregnancies. Am J Reprod Immunol 2014, 72:422e434 Bachmayer N, Rafik Hamad R, Liszka L, Bremme K, SverremarkEkström E: Aberrant uterine natural killer (NK)-cell expression and altered placental and serum levels of the NK-cell promoting cytokine interleukin-12 in pre-eclampsia. Am J Reprod Immunol 2006, 56: 292e301 de Moraes-Pinto MI, Vince GS, Flanagan BF, Hart CA, Johnson PM: Localization of IL-4 and IL-4 receptors in the human term placenta, decidua and amniochorionic membranes. Immunology 1997, 90: 87e94 Myerson D, Parkin RK, Benirschke K, Tschetter CN, Hyde SR: The pathogenesis of villitis of unknown etiology: analysis with a new conjoint immunohistochemistry-in situ hybridization procedure to
ajp.amjpathol.org
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
9
1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116
1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132Q14 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178
Derricott et al
25.
26. 27. 28.
29.
30.
31. 32.
33.
34. 35. 36.
10
identify specific maternal and fetal cells. Pediatr Dev Pathol 2006, 9: 257e265 Labarrere C, Faulk W, McIntyre J: Villitis in normal term human placentae: frequency of the lesion determined by monoclonal antibody to HLA-DR antigen. J Reprod Immunol 1989, 16:127e135 Krieger NR, Yin DP, Fathman CG: CD4þ but not CD8þ cells are essential for allorejection. J Exp Med 1996, 184:2013e2018 Chaplin DD: Overview of the immune response. J Allergy Clin Immunol 2010, 125(2 Suppl 2):S3eS23 Feeley L, Mooney EE: Villitis of unknown aetiology: correlation of recurrence with clinical outcome. J Obstet Gynaecol 2010, 30: 476e479 Ito Y, Matsuoka K, Uesato T, Sago H, Okamoto A, Nakazawa A, Hata K: Increased expression of perforin, granzyme B, and C5b-9 in villitis of unknown etiology. Placenta 2015, 36:531e537 Medawar PB: Some immunological and endocrinological problems raised by evolution of viviparity in vertebrates. Symp Soc Exp Biol 1953, 7:320e328 Challis JR, Lockwood CJ, Myatt L, Norman JE, Strauss JF 3rd, Petraglia F: Inflammation and pregnancy. Reprod Sci 2009, 16:206e215 Raghupathy R, Makhseed M, Azizieh F, Hassan N, Al-Azemi M, AlShamali E: Maternal Th1- and Th2-type reactivity to placental antigens in normal human pregnancy and unexplained recurrent spontaneous abortions. Cell Immunol 1999, 196:122e130 Cousins DJ, Lee TH, Staynov DZ: Cytokine coexpression during human Th1/Th2 cell differentiation: direct evidence for coordinated expression of Th2 cytokines. J Immunol 2002, 169:2498e2506 Perricone C, de Carolis C, Perricone R: Pregnancy and autoimmunity: a common problem. Best Pract Res Clin Rheumatol 2012, 26:47e60 Jamieson DJ, Theiler RN, Rasmussen SA: Emerging infections and pregnancy. Emerg Infect Dis 2006, 12:1638e1643 Lombardelli L, Aguerre-Girr M, Logiodice F, Kullolli O, Casart Y, Polgar B, Berrebi A, Romagnani S, Maggi E, Le Bouteiller P,
37.
38.
39.
40.
41.
42.
43. 44.
45.
Piccinni M-P: HLA-G5 induces IL-4 secretion critical for successful pregnancy through differential expression of ILT2 receptor on decidual CD4þ T cells and macrophages. J Immunol 2013, 191:3651e3662 Alijotas-Reig J, Llurba E, Gris JM: Potentiating maternal immune tolerance in pregnancy: a new challenging role for regulatory T cells. Placenta 2014, 35:241e248 Ingman K, Cookson VJ, Jones CJP, Aplin JD: Characterisation of Hofbauer cells in first and second trimester placenta: incidence, phenotype, survival in vitro and motility. Placenta 2010, 31:535e544 Burton GJ, Kaufmann P, Huppertz B: Anatomy and genesis of the placenta. Edited by Neill JD. The Physiology of Reproduction. ed 3. St. Louis, Elsevier, 2006, pp 211e266 Mullins E, Prior T, Roberts I, Kumar S: Changes in the maternal cytokine profile in pregnancies complicated by fetal growth restriction. Am J Reprod Immunol 2012, 68:1e7 Mullins E, Prior T, Roberts I, Kumar S: Changes in the fetal and neonatal cytokine profile in pregnancies complicated by fetal growth restriction. Am J Reprod Immunol 2013, 69:441e448 Young OM, Tang Z, Niven-Fairchild T, Tadesse S, Krikun G, Norwitz ER, Mor G, Abrahams VM, Guller S: Toll-like receptormediated responses by placental Hofbauer cells (HBCs): a potential pro-inflammatory role for fetal M2 macrophages. Am J Reprod Immunol 2015, 73:22e35 Altemani A, Gonzatti A, Metze K: How many paraffin blocks are necessary to detect villitis? Placenta 2003, 24:116e117 Barnes DM, Harris WH, Smith P, Millis RR, Rubens RD: Immunohistochemical determination of oestrogen receptor: comparison of different methods of assessment of staining and correlation with clinical outcome of breast cancer patients. Br J Cancer 1996, 74: 1445e1451 Khong TY: Expression of MHC class II antigens by placental villi: no relationship with villitis of unknown origin. J Clin Pathol 1995, 48: 494e495
ajp.amjpathol.org
-
The American Journal of Pathology
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240
1241 1242 1243 1244 1245 1246 1247
Supplemental Figure S1
Representative images of placental sections immunostained for cytokines. A, C, and D: VUE lesions were stained for IL-10 (A), IL-6 (C), and TGF-b (D). B: Cytokine staining of a placental section from NP immunostained for IL-10. Scale bar Z 50 mm. Original magnification, 100. NP, normal pregnancy; TGF, transforming growth factor; VUE, villitis of unknown etiology.
Q13
Supplemental Figure S2 Quantification of cytokine staining intensity using HistoQuest image analysis software. IL-2 (A), IL-12 (B), IL-4 (C), and IL-10 (D). Horizontal lines represent median. *P < 0.05, **P < 0.01, and ***P < 0.001 by Kruskal-Wallis test with Dunn’s post hoc test. DAB, diaminobenzidine; NP, normal pregnancy; SB FGR, stillbirth with fetal growth restriction; LB FGR, live birth with fetal growth restriction.
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
1248 1249 1250 1251 1252 1253 1254
The American Journal of Pathology, Vol. -, No. -, - 2016
ajp.amjpathol.org
Characterizing Villitis of Unknown Etiology and Inflammation in Stillbirth Q24
Hayley Derricott,* Rebecca L. Jones,* Susan L. Greenwood,* Gauri Batra,y Margaret J. Evans,z and Alexander E.P. Heazell* From the Maternal and Fetal Health Research Centre,* Institute of Human Development, University of Manchester, Manchester Academic Health Science Centre, Manchester; the Department of Paediatric Histopathology,y Royal Manchester Children’s Hospital, Manchester; and the Department of Paediatric Histopathology,z Edinburgh Royal Infirmary, Edinburgh, United Kingdom Accepted for publication December 8, 2015.
Q3
Q5
Address correspondence to Hayley Derricott, Maternal & Fetal Health Research Centre, 5th Floor St. Mary’s Hospital, Oxford Rd., Manchester M13 9WL, United Kingdom. E-mail: hayley.derricott@ manchester.ac.uk.
Villitis of unknown etiology (VUE) is an enigmatic inflammatory condition of the placenta associated with fetal growth restriction and stillbirth. Greater understanding of this condition is essential to understand how it contributes to adverse outcomes. Our aim was to identify and quantify the cells in VUE in cases of stillbirth and to characterize immune responses specific to this condition. Immunohistochemistry was performed on placentas from stillborn infants whose cause of death was recorded as VUE to identify CD45þ leukocytes, CD163þ macrophages, CD4þ T cells, CD8þ T cells, neutrophils, and proinflammatory and antiinflammatory cytokines. Images were quantified with HistoQuest software. CD45þ leukocytes comprised 25% of cells in VUE lesions: macrophages (12%) and CD4 T cells (11%) being the predominant cell types; CD8 T cells were also observed in all lesions. Leukocytes and macrophages were increased throughout the placenta in stillbirths; pan-placental CD4þ and CD8þ T cells outside VUE lesions increased in stillbirth with VUE. Increased IL-2 and IL-12 and reduced IL-4 immunostaining was seen in VUE lesions. Our results suggest VUE in stillbirth has a similar immune cell profile to live birth. Pan-placental macrophages, CD4 and CD8 T cells indicate a wider inflammatory response unrestricted to VUE lesions. The cytokine profile observed suggests a skew toward inappropriate Th1 immune responses. Full characterization of VUE lesion phenotype confirms its immunologic origins and provides foundations to develop novel investigations. (Am J Pathol 2016, -: 1e10; http://dx.doi.org/10.1016/j.ajpath.2015.12.010)
Stillbirth, the death of a baby after 20 weeks’ gestation, places a sustained economic and emotional burden on both maternity services and the wider community. Despite significant effort, the stillbirth rate has decreased at a slower rate than neonatal or infant death.1 In high-income countries stillbirth rates are variable. The United States and United Kingdom perform relatively poorly with rates of 1 in 160 and 1 in 212, respectively, whereas in Finland 1 in 400 pregnancies result in a stillborn infant.2e4 Placental dysfunction as a cause of stillbirth has increasingly become a focus of research effort with recognition that placental disorders are the cause of death in up to 65% of stillbirths.5 However, the placental lesions associated with stillbirth are extremely varied and poorly defined, and their relation to pathologic processes leading to stillbirth is difficult to assess.6,7 Placental lesions associated with stillbirth include those with genetic, environmental, infective, inflammatory, mechanical, metabolic, and vascular origins.6
Understanding of inflammatory disorders of the placenta remains relatively limited in comparison with vascular lesions. Two main inflammatory conditions have been linked to placental dysfunction in the absence of infection: villitis of unknown etiology (VUE) and chronic histiocytic intervillositis.8 VUE is an inflammatory condition of the placenta reported to occur in up to 15% of term placentas and more frequently in pregnancies resulting in poor outcome.9,10 The origin of VUE is unclear, although it is proposed to be maternal immune rejection of a semiallogeneic placenta.11e13 VUE is identified and characterized by the presence of elevated numbers of fetal macrophages (Hofbauer cells) and an infiltrate of maternal T lymphocytes in the villous stroma. Areas of the placenta not affected by VUE lesions display apparently normal structural characteristics.9 The cell profile of VUE has Supported by Tommy’sethe Baby Charity (H.D.). Disclosures: This work forms part of a Ph.D. project for H.D.
Copyright ª 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2015.12.010
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 Q4 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 Q1 Q2 123 124
125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186
Derricott et al Table 1
Antibodies and Positive Control Tissues Used for Identifying Immune Cell Populations and Cytokines in Placental Villous Tissue
Antigen
Antigen retrieval
Supplier
Working concentration (mg/mL)
Ab type
Control tissue
CD45 CD163 CD4 (clone 4B12) CD8 NE IL-2 IL-4 IL-6 IL-10 IL-12 TGF-b
Sodium citrate Sodium citrate Tris-EDTA or sodium citrate Tris-EDTA None Sodium citrate Sodium citrate Sodium citrate None Sodium citrate Sodium citrate
Dako AbD Serotec Dako Dako Dako Novus Biologicals Thermo Scientific Novus Biologicals Abcam Sigma-Aldrich Novus Biologicals
0.5 10 4.6 1.6 1.1 4.5 2.5 9.6 5 2.5 10
M mAb M mAb M mAb M mAb M mAb R pAb R pAb M mAb M mAb R pAb M mAb
Tonsil Tonsil Tonsil Tonsil PTLI Tonsil Tonsil Tonsil Liver (Kupffer cells) Trophoblast Tonsil
M mAb, mouse monoclonal antibody; NE, neutrophil elastase; PTLI, preterm labor with infection; R pAb, rabbit polyclonal antibody; TGF, transforming growth factor.
previously been characterized in both appropriately grown and growth-restricted live born infants.12,14e16 Conflicting reports state either CD4þ or CD8þ T cells are the most common lymphocyte type in VUE lesions.12,14,17,18 The subtype of CD4þ cells (Th1/Th2) has not been examined; therefore, the type of immune response in VUE remains to be elucidated. A thorough characterization of VUE has yet to be completed in stillbirth; it is not understood why it may cause fetal demise. In addition, the cytokine profile in VUE has not been described in either live births or stillbirths. Characterization of the cell types present and their cytokine profile will provide a greater understanding of the pathophysiology of VUE. We hypothesized that the immune cells in VUE lesions in stillbirth would be comparable with those described in live birth and that evidence of widespread inflammatory changes would be detectable in the placentas of stillborn infants with VUE. In this immunomorphologic study we aimed to accurately quantify the type and number of immune cells present in VUE lesions, assess leukocyte infiltration across the placenta, and analyze the expression of a range of proinflammatory and anti-inflammatory cytokines with the use of immunohistochemical approaches, coupled with an unbiased sensitive image analysis method. We compared leukocyte infiltration and cytokine expression across the placenta to ascertain whether this was altered in Table 2
dysfunctional placentas from stillbirth with and without VUE and fetal growth restriction (FGR) compared with healthy controls.
Materials and Methods Study Population Placental tissues from pregnancies resulting in stillbirth in which a contributory cause of death was recorded as VUE (n Z 12) or FGR (n Z 12) were obtained from the Pediatric Histopathology departments of Manchester Royal Children’s Hospital and Edinburgh Royal Infirmary. Samples from stillborn growth-restricted infants had no evidence of VUE lesions recorded on postmortem examination. Consent for tissue use in research was obtained at the time of consent for postmortem; their use in this project was approved by Humber Bridge Research Ethics Committee (Ref. 13/YH/0176). Live born infants (n Z 11) with an individualized birth weight centile less than the fifth were considered FGR infants; placental tissue was obtained from the Maternal and Fetal Health Research Group Biobank, University of Manchester (North West REC 08/H1010/55). Matched placental samples from women with uncomplicated pregnancies (n Z 12) were selected from the Maternal and Fetal Health Research
Demographic Information for the Women Included in the Study
Characteristic
Stillbirth with VUE (n Z 12)
Stillbirth with FGR (n Z 12)
Live birth with FGR (n Z 11)
Normal pregnancy (n Z 12)
P
Maternal age (years) Gestational age (weeks) Gravidity Parity Birthweight (g) IBC
32 (21e36) 39 (20e41) 3 (1e7) (n Z 7) 1 (0e3) (n Z 7) NA 23 (3e90) (n Z 7)
23.5 35.21 1 0 1061 NA
32 37.8 1 0 2246 3
35 33.5 2 1 3430 45
0.019 0.098 0.298 0.118 <0.001 <0.001
(19e42)* (28e38) (1e4) (0e2) (385e2000)***
(16e35) (27.7e42.1) (1e9) (0e3) (747e3060)** (0e10)***
(25e42) (30.0e41.7) (1e18) (0e3) (2900e3840) (12e93)
Results are presented as median (range). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with normal pregnancy. FGR, fetal growth restriction; IBC, individualized birth weight centile; NA, not available; VUE, Villitis of unknown etiology.
2
ajp.amjpathol.org
-
The American Journal of Pathology
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
Q23
187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248
extensive CD45þ cell infiltrate that displayed the morphologic properties of macrophages and lymphocytes. Subsequent immunostaining for specific immune cells was performed to identify CD4þ T cells, CD8þ T cells, CD163þ macrophages, and neutrophils. Further immunostaining for cytokines IL-2, IL-4, IL-10, and IL-12 was performed on representative sections from all samples. Finally, staining for IL-6 and transforming growth factor (TGF)-b was performed on selected stillbirth samples with VUE (n Z 6) and healthy pregnancies (n Z 6). Heat-mediated antigen retrieval was performed for all antibodies except antieneutrophil elastase and antieIL-10 monoclonal antibodies. Primary antibodies were applied at the working concentrations detailed in Table 1. ½T1 Each tissue section had a corresponding negative control on which mouse or rabbit IgG (Sigma-Aldrich) was applied at the Q8 same concentration as the primary antibody. Each staining run included a positive control tissue as described in Table 1.
print & web 4C=FPO
249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310
Characterizing VUE in Stillbirth
Q15
Q16
Immunofluorescence
Figure 1 Serial images of a representative VUE lesion immunostained for immune cell markers. CD45 (A), CD163þ macrophages (B), CD8 cytotoxic T cells (C), CD4þ T helper cells (D), neutrophil elastase (E), and negative (F). Arrows highlight examples of positively stained cells. Scale bar Z 50 mm. Original magnification, 100. VUE, villitis of unknown etiology.
Group Biobank and used as controls. All placentas were taken from mothers who showed no evidence of conditions such as preeclampsia or diabetes. Detailed case note review showed no clinical, biochemical, hematologic, or microbiological evidence of infection was present. Multiple pregnancies, abnormal fetal heartbeat (live birth only), and fetuses with anomalies were excluded. Q6
Sample Collection Placentas from cases of stillbirth had two to seven randomly selected samples per placenta obtained at the time of postmortem for histologic analysis. For live births three samples of placental villous tissue were taken, one each from the center, middle, and periphery of the tissue.19 Biopsies were fixed in neutral-buffered formalin for 24 hours before being processed and embedded in paraffin wax.
IHC
Q7
For immunohistochemistry (IHC), 5-mm serial sections were cut and mounted onto slides coated with poly-L-lysine (Sigma-Aldrich, Poole, UK). Immunostaining with colorimetric detection was performed as described previously.20 VUE lesions were initially identified by detecting clusters of leukocytes with the use of a mouse monoclonal antibody to CD45 (leukocyte common antigen). A VUE lesion was defined as an area of villous tissue with evidence of an
The American Journal of Pathology
-
The phenotype of the macrophages and T cells in the placentas/ lesions was investigated by dual immunofluorescence with combinations of immune cell markers CD4 and CD163 and cytokines IL-2, IL-12, or IL-4. Sections were fixed and processed as described in the previous section. Autofluorescence was quenched by 1-hour incubation in 0.25% ammonia (SigmaAldrich) in 70% ethanol. Primary antibodies were applied at the concentrations described in Table 1 at 4 C overnight. Secondary antibodies (goat anti-mouse Alexa 568 or goat antirabbit Alexa 488; Life Technologies, Paisley, UK) were applied at 5 mg/mL and incubated for 30 minutes at room temperature. DAPI-containing aqueous mounting medium was used to apply coverslips.
Image Capture and Analysis Tissue sections were visualized with the 10 objective of an Olympus BX41 light microscope (Southend-on-Sea, UK). Images were captured with QIcam Fast 1394 camera (QImaging, Surrey, BC, Canada) and Image Pro Plus 6.0 (Media Cybernetics Inc., Rockville, MD). Quantitative, unbiased image analysis was completed with HistoQuest image analysis software (TissueGnostics, Vienna, Austria). The Q9 pixels of images viewed in this software are converted to grayscale (0 to 256 scale) and assigned an arbitrary number relating to staining intensity. The image analysis software differentiates hematoxylinþ and diaminobenzidine (DAB)þ Q10 cells or areas that are recorded as events (cells/nuclei) or area of staining. Results are presented as histograms or dot plot scattergrams to which staining area and/or intensity cutoffs can be applied to differentiate between cell populations. Analysis variables were optimized such that the number of DABþ events and hematoxylinþ events were counted. Two analyses were performed. First, for characterization of VUE lesions in stillbirth, inflammatory foci were identified in CD45-stained sections, then the remaining immunostains were
ajp.amjpathol.org
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
3
311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372
Figure 2 AeC: Screenshots from HistoQuest image analysis software demonstrating the selection of DABþ events in sections stained for CD45 leukocyte common antigen (A and B) and cytotoxic T-lymphocyte marker CD8 (C). Positive events are highlighted in red; events encircled in green indicate hematoxylin positive/DAB events. D: A representative scattergram from which staining intensity variables were ascertained. Scale bar Z 50 mm. Original magnification, 100. DAB, diaminobenzidine.
print & web 4C=FPO
373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434
Derricott et al
Q11
Q17 Q18
Q19
matched and captured in serial sections. In tissue sections with widespread/numerous inflammatory foci, representative lesions were selected (n Z 2 to 5 per section). To assess pan-placental inflammation in tissue, 10 random images (excluding lesions) were taken for each placental slide from all groups (stillbirth with VUE, stillbirth with FGR, live birth with FGR, and controls), immunostained for CD45, CD163 macrophage, CD4 T cell, and CD8 T cell markers. DABþ cells and nuclei were quantified to ascertain whether there were any differences in leukocyte numbers between the study groups. The presence of cytokines was determined by immunostaining for IL-2, IL-4, IL-6, IL-10, IL-12, and TGF-b. Images were analyzed quantitatively in sections of tissue from VUE lesions, areas of stillbirth with VUE placentas that were devoid of lesions, stillbirth with FGR placentas, live birth with FGR placentas, and controls (n Z 10 images per placenta). Area and intensity of staining were recorded for each placental sample, and quantitative analysis was completed with HistoQuest image analysis software. Fluorescent-stained sections were visualized with a Zeiss fluorescence microscope, AxioCam MRn camera with Zen pro image processing software (Zeiss, Welwyn Garden City, UK).
(GraphPad Software, San Diego, CA). Nonparametric categorical data were analyzed with Fisher’s exact test; nonparametric continuous data were analyzed with U test or Kruskal-Wallis test with Dunn’s post hoc test.
Statistical Analysis
Characterization of Focal Inflammatory Lesions
We powered our analysis to determine the number of CD45þ and CD163þ cells in placentas from stillborn infants, assuming similar increases in these cells as seen in placentas from poor outcome pregnancies experiencing reduced fetal movements.21 No similar studies have been conducted with CD4 and CD8 T cells, so we were not able to take direct comparisons. For 80% power with an a value of 0.05, the sample size was calculated as 12. All statistical analysis was performed with GraphPad Prism software version 6.0
VUE lesions were identified in all placentas from the stillbirth with VUE cohort; a representative lesion is shown in Figure 1. In total 104 VUE lesions from 12 placentas were ½F1 photographed and analyzed. The number of CD45þ cells in the lesions contributed 25% (median; range, 8% to 48%) to the total number of nuclei. Macrophages and CD4þ T lymphocytes were the most prevalent immune cells in the lesions, accounting for 12% (9% to 23%) and 11% (3% to 14%) of the total number of nuclei, respectively. CD8þ T
4
Results Participant Demographic Characteristics The demographic data for the 48 women included in the study are shown in Table 2. A significant difference was found in ½T2 maternal age between the stillbirth with FGR and normal pregnancy groups (P Z 0.02). Contributory causes of death for the stillborn infant cohorts are detailed in Supplemental Table S1. By experimental design birth weight and individualized birth weight centile differences between stillbirth with FGR, live birth with FGR, and normal pregnancies were highly significant (P < 0.001 and P < 0.01, respectively). Average time between fetal death and delivery of infant was 3.5 days (range, 1 to 14 days). Comparisons of the grading/ extent of VUE between tissue samples could not be drawn because this information was not recorded in postmortem reports (Supplemental Table S2).
ajp.amjpathol.org
-
The American Journal of Pathology
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496
lymphocytes were also present in VUE lesions, contributing 4% (0.3% to 11%) to the total number of nuclei. Neutrophil levels were low in all samples, accounting for 1% (0.37% to 2%) of total number of nuclei.
Differentiating Cell Populations Cell populations were differentiated by applying staining area and/or intensity cutoffs to the results presented as his½F2 tograms or dot plot scattergrams (Figure 2).
print & web 4C=FPO
497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558
Characterizing VUE in Stillbirth
The American Journal of Pathology
-
559 560 561 562 563 564 565 566 Figure 3 Quantification of pan-placental im567 mune cells in normal and pathologic pregnancies. 568 CD45 (A), macrophages (CD163, B), cytotoxic 569 T cells (CD8, C), and T helper cells (CD4; D). 570 Horizontal lines represent median. *P < 0.05, **P < 0.01, and ***P < 0.001, Kruskal-Wallis test 571 with Dunn’s post hoc test. LB FGR, live birth with 572 fetal growth restriction; NP, normal pregnancy; SB 573 FGR, stillbirth with fetal growth restriction; SB 574 VUE, stillbirth with villitis of unknown etiology. 575 576 577 578 579 580 581 582 583 584 Quantification of Pan-Placental Immune Cells 585 586 Significant increases were detected in the number of pan587 placental CD45þ leukocytes in all placentas from pregnancies 588 from stillborn infants (with or without VUE) compared with 589 normal pregnancies (P < 0.001) (Figure 3A). Significant ½F3 590 elevation in macrophage numbers was detected in the pla591 centas from stillborn infants with VUE (P < 0.01), stillborn 592 593 infants with FGR (P < 0.001), and live born infants with FGR 594 (P < 0.01) compared with normal pregnancy (Figure 3B). 595 Placentas from the stillbirth with VUE group showed 596 597 598 599 600 601 602 Figure 4 Representative images of placental 603 sections immunostained for proinflammatory and 604 anti-inflammatory cytokines. A, D, and G: VUE lesions were stained for IL-2 (A), IL-12 (D), and IL-4 605 (G). B, E, and H: Cytokine staining in areas of 606 placenta unaffected by lesions from stillborn in607 fants with VUE were stained for IL-2 (B), IL-12 (E), 608 and IL-4 (H). C, F, and I: Representative sections 609 of placenta from pregnancies with a normal 610 outcome were stained for IL-2 (C), IL-12 (F), and 611 Q20 IL-4 (insets, negatives) (I). Arrows highlight 612 staining associated with cells; arrowheads high613 light staining associated with trophoblast. Scale 614 bar Z 50 mm. Original magnification, 100. VUE, villitis of unknown etiology. 615 616 617 618 619 620
ajp.amjpathol.org
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
5
Derricott et al
print & web 4C=FPO
621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 Figure 5 Representative high power images of placental sections after 646 immunostaining for proinflammatory and anti-inflammatory cytokines. 647 A, C, and E: VUE lesions were stained for IL-2 (A), IL-12 (C), and IL-4 (E). B, D, and F: Representative sections of placenta from pregnancies with a 648 normal outcome were stained for IL-2 (B), IL-12 (D), and IL-4 (F). Arrows 649 highlight staining of cells in the stromal core, arrowheads highlight 650 staining associated with syncytiotrophoblast. Scale bar Z 20 mm. Original 651 magnification, 400. VUE, villitis of unknown etiology. 652 653 654 significantly more pan-placental CD4 and CD8 cells than all 655 other groups (P < 0.001) and were observed in every field of 656 view, excluding lesions (Figure 3, C and D). 657 658 659 Cytokines 660 661 To fully characterize the immune response in VUE, im662 munostaining of characteristic Th1 (IL-2, IL-12) and Th2 663 (IL-4) cytokines was performed (Figure 4). Staining of IL-2 ½F4 664 and IL-12 localized to cells within the stromal core and 665 stillborn, as previously shown22 (Figure 5). Area of IL-12 ½F5 666 667 staining was significantly increased in VUE lesions and 668 live born FGR placentas compared with normal pregnancy 669 (P < 0.001). A significant increase in area of IL-2 staining 670 ½F6 was observed in VUE lesions (P < 0.05) (Figure 6). IL-4 671 staining localized to both stromal and trophoblast cells23 672 (Figure 5). A significant reduction was found in the area 673 of IL-4 immunostaining in VUE lesions and areas devoid of 674 lesions compared with controls (P < 0.05 and P < 0.01, 675 respectively). A significant reduction in IL-4 staining area 676 was also observed in the stillbirth with FGR group 677 compared with live birth with FGR and controls (P < 0.05 678 679 and P < 0.001, respectively). IL-10 appeared significantly 680 increased overall in areas of VUE placentas devoid of 681 lesions (P < 0.001), but individual comparisons failed to 682
6
reach statistical significance (Figures 4 and 6, Supplemental Figure S1). Staining intensity for all cytokines was significantly different between samples (Supplemental Figure S2). Intensity of IL-2 was significantly reduced (stillbirth FGR, P Z 0.006; live birth FGR, P Z 0.0004) and IL-12 increased (stillbirth FGR, P Z 0.0002; live birth FGR, P < 0.001) in FGR samples compared with control. IL-4 intensity was significantly reduced in VUE lesions compared with control (P Z 0.02). IL-10 staining intensity was significantly increased in VUE placentas compared with stillbirth with FGR (VUE lesion, P Z 0.03; VUE nonlesion, P Z 0.01). Immunostaining for cytokines IL-6 and TGF-b was not detected in VUE lesions (Supplemental Figure S1).
Macrophage and T-Cell Phenotype Dual immunofluorescence was performed to identify immune cell sources of cytokines of interest. IL-2 co-localized with CD4þ staining, CD163þ on the cell surface co-localized with cytoplasmic IL-12 staining, and IL-4 co-localized with CD4þ (Figure 7). Evidence of faint IL-4 fluorescence was found in ½F7 the syncytiotrophoblast layer as observed in the images of chromogen staining (Figures 4 and 7).
Discussion This study has fully characterized VUE in placentas from stillborn infants by quantifying the immune cells found in the lesions by using unbiased image analysis software, after IHC to detect specific immune cell types. We have also demonstrated specific pan-placental inflammatory changes in stillbirths (with and without VUE), indicating a more widespread immune reaction occurring with localized VUE lesions. Furthermore, our cytokine analyses indicate that the immune response in the lesions is directed toward Th1-type response. Previous studies of VUE in live birth have described lesions comprising mainly fetal macrophages24 and maternal CD4þ18,25 or CD8þ12,14 T lymphocytes. Our results support these descriptions: in cases of stillbirth, macrophages and CD4þ T cells predominate, although elevated numbers of CD8 cells are also present. A key strength of the current study is the use of immunostaining to detect specific immune cell types. Pathologists examining hematoxylin and eosin-stained sections would not be able to identify individual leukocyte subsets, which are clearly visible after immunostaining for leukocyte, macrophage, CD4, and CD8 cell markers. VUE has been likened to graft rejection or graft-versus-host disease.11e13 The phenotype that we observed, with CD4þ lymphocytes outnumbering CD8þ lymphocytes, agrees with the 1989 study of Labarrere et al25 and supports this hypothesis because the T-helper cells would assume responsibility for directing the immune response toward a Th1-type response.26,27 The presence of cytotoxic CD8 T cells in the lesions may contribute to both
ajp.amjpathol.org
-
The American Journal of Pathology
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744
745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806
Characterizing VUE in Stillbirth
Figure 6 Quantification of cytokine staining area using HistoQuest image analysis software. AeD: IL-2 (A), IL-12 (B), IL-4 (C), and IL-10 (D). Horizontal lines represent median. *P < 0.05, **P < 0.01, and ***P < 0.001, Kruskal-Wallis test with Dunn’s post hoc test. DAB, diaminobenzidine; LB FGR, live birth with fetal growth restriction; NP, normal pregnancy; SB FGR, stillbirth with fetal growth restriction; VUE, villitis of unknown etiology.
the fetal placental vasculopathy often observed in VUE and the recently described elevated apoptosis of intravillous cells.9,17,28,29 Since Medawar30 first described the immunologic paradox of pregnancy, much has been published on the shift from Th1 to Th2 responses in pregnancy.31e35 Suppression of antiefetal cell-mediated responses and a skew toward antibody-mediated immunity appears to be advantageous in the maintenance of a successful pregnancy.35,36 The cytokine profile differs between Th1 and Th2 responses; IL-2, IL-6, and IL-12 are associated with Th1 responses, whereas anti-inflammatory cytokines IL-4, IL-10, and TGF-b predominate in Th2-type responses. Our findings of increased levels of IL-2 and IL-12 in VUE lesions suggest that there may be an inappropriate shift toward cell-mediated immunity in VUE cases associated with stillbirth. Staining intensity of IL-2 and IL-12 was not significantly different between VUE samples and controls, implying that more cells are secreting the cytokine but the expression of it is not different. Co-localized immunofluorenscence staining of CD163/IL-12 and CD4/IL-2 is indicative of proinflammatory macrophage and Th1 CD4 T-cell phenotypes. We have also detected a significant reduction in levels of anti-inflammatory IL-4 in VUE placentas from cases of stillbirth. The reduction in IL-4 levels was mirrored by a significant reduction in expression in VUE lesions. The placental anti-inflammatory cytokine environment characterized by IL-4, IL-10, and TGF-b, which induces maternal tolerance of the fetus, is
The American Journal of Pathology
-
critical in maintaining a healthy pregnancy.32,36,37 Conversely, levels of proinflammatory IL-12 remain low throughout successful pregnancy.36 Significant decreases in IL-4 in stillbirth placentas may be a contributory factor in permitting a shift from Th2-type to Th1-type immunity. These data support a reduction in Th2 and a Th1 bias in VUE lesions, which may lead to unresolved cell-mediated inflammation. Therefore, the critical balance appears to be disrupted in VUE lesions in cases ending in stillbirth. It has been stated that macrophages and their cytokines are fundamental in establishing and maintaining the placenta at the start of pregnancy.38,39 It is therefore reasonable to suggest that an excess of immune cells and proinflammatory cytokines in late pregnancy may have a detrimental effect on placental function, which is then manifested as FGR or stillbirth. These effects would be in addition to the cytotoxic effects of any immune response mounted. Interestingly, systematic reviews suggest a general shift toward a proinflammatory cytokine profile in maternal serum and placentas from FGR and small for gestational age infants, confirming a role for a proinflammatory state in FGR.40,41 Conflicting reports of M2 placental macrophages potentially acting as either proinflammatory42 or immunosuppressive29 cells only serve to compound the mystery underlying VUE. Our investigation has shown placental macrophages expressing the M2 marker CD163, yet producing the proinflammatory cytokine IL-12, further enhancing the evidence of the
ajp.amjpathol.org
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
7
Q21
807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868
Figure 7
Representative high power images of placental sections after dual immunofluorescence for proinflammatory and anti-inflammatory cytokines and cell markers. AeC: VUE lesions stained for CD4 (red), IL-2 (green), co-localization (yellow) (A), CD163 (red) and IL-12 (green) (B), and CD4 (red) and IL-4 (green), co-localization (yellow) (C). D: Mouse and rabbit IgG isotype control. Red arrows highlight dual staining; yellow arrows indicate co-localized immunofluorescence; and white arrowheads indicate trophoblast-localized IL-4. Scale bar Z 20 mm. Original magnification, 630. VUE, Villitis of unknown etiology.
print & web 4C=FPO
869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930
Derricott et al
Q12
plasticity of these cells and their potential role in pregnancy complications. Pan-placental increases in leukocyte numbers in VUE are a novel finding. Significantly elevated numbers of leukocytes and macrophages were detected in all stillbirths compared with controls. The elevation in macrophages in live born FGR suggests that this is not a postmortem artifact. These data are consistent with our recent findings of elevated placental macrophage numbers in pregnancies at high risk of stillbirth.21 Staining intensity of cytokines in both live born and stillborn FGR were consistent throughout, further confirming that the structural characteristics observed is not related to postmortem tissue changes. The placentas from these pregnancies also exhibited a proinflammatory bias in relation to cytokine mRNA and protein expression (elevated IL-1 family members, reduced IL-10 and IL-4). Together, these data are highly suggestive of placental inflammation being an underlying factor in pregnancies that result in FGR and stillbirth. Previous descriptions of VUE have stated the occurrence of focal villous lesions with adjacent unaffected tissue displaying normal structural characteristics.9 It has been suggested that the occurrence of VUE may have been underestimated because sampling of the placenta was not extensive enough to find evidence of lesions43; Altemani et al43 reported that at least six blocks are needed to achieve an 85% chance of diagnosing 95% of cases. In our study, CD4 and CD8 T-cell numbers were increased in stillbirths with VUE compared with all other group samples, and these increases were not confined to VUE lesions. This finding is a strength of the specificity of the IHC in our study. A sensitive and unbiased image analysis tool that can accurately detect immunostained cells with the use of a relatively low magnification (100) also contributes to the strength of the study. Our data suggest that a pan-placental increase in
8
CD4 and CD8 T cells should stimulate the pathologist to suspect an inflammatory condition, such as VUE, without relying on locating a specific lesion. With the use of IHC and sensitive image analysis software such as HistoQuest, the incidence of VUE could potentially be identified and accurately reported with fewer tissue samples. A general consensus on the sampling and definitions of placental lesions, as discussed by the Amsterdam Placental Workshop Group, attempts to address issues such as this (T.V. Khong et al, unpublished data). More sophisticated placental pathologic investigations may improve diagnosis of VUE (as was the case with estrogen receptor expression in breast cancer44) and thus provide much sought after explanations for the cause of death. By examining placental tissue after delivery, IHC can only provide a snapshot of VUE, rather than a temporal profile of the cell migration and its associated effects. This could explain why descriptions of the proportions of T cells in VUE differ between research studies because they sample different periods in the evolution of lesions.12,14,17,18 In addition, the panplacental presence of CD4 and CD8 T cells may represent pre-lesions that, given time, would expand within the villous stroma, potentially explaining why VUE is considered a thirdtrimester phenomenon.9 The 1995 study by Khong,45 describing expression of major histocompatibility complex type II in both nonvillitis and villitis-affected placentas, also suggests that VUE may be an evolving lesion. This dynamic process cannot be surmised from the current method of studying placental tissue. The results of this study would be further enhanced by including samples of placenta from live born infants with evidence of VUE. At the time the study was conducted, these were not available. A greater understanding of the immune responses associated with VUE may lead to the development of screening
ajp.amjpathol.org
-
The American Journal of Pathology
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
Q22
931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992
993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054
Characterizing VUE in Stillbirth tests to enable antenatal detection of VUE. Our laboratory recently published data that indicate alterations in concentrations of maternal serum cytokines in pregnancies characterized by placental inflammation.21 This is supported by the observation of elevated maternal plasma chemokines (CXCL9, CXCL10, and CXCL11) in cases of VUE.13 It may therefore be possible to identify placental inflammation antenatally which would allow increased antenatal surveillance and potential anti-inflammatory therapies to prevent stillbirth.
7.
8. 9. 10.
11.
Conclusion We have fully characterized VUE in stillbirths with the use of IHC to identify specific immune cell types and have detected evidence of a Th1-type cytokine response. These data present novel insights into the pathogenesis of VUE and provide solid foundations for functional studies to determine the impact of inflammation on placental function.
12.
13.
Acknowledgments 14.
We thank the mothers who gave consent to use the placentas that we studied, the recruiters, Dr. Mark Wareing who coordinated the Maternal and Fetal Health Research Group Biobank, Dr. Sylvie Girard for allowing us to use some of the immunostained sections from healthy pregnancies, Dr. Rachael Wright who dedicated a lot of time optimizing antibodies for IHC, and Dr. Joanne Pennock for immunologic advice.
15.
16.
17.
Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2015.12.010.
18.
19.
References 1. Flenady V, Middleton P, Smith GC, Duke W, Erwich JJ, Khong TY, Neilson J, Ezzati M, Koopmans L, Ellwood D, Fretts R, Frøen JF; Lancet’s Stillbirths Series steering committee: Stillbirths: the way forward in high-income countries. Lancet 2011, 377:1703e1717 2. MacDorman M, Kirmeyer S: Fetal and perinatal mortality, United States, 2005. Natl Vital Stat Rep 2009, 57:1e19 3. Lawn JE, Blencowe H, Pattinson R, Cousens S, Kumar R, Ibiebele I, Gardosi J, Day LT, Stanton C; Lancet’s Stilbirths Series steering committee: Stillbirths: Where? When? Why? How to make the data count? Lancet 2011, 377:1448e1463 4. Office of National Statistics: Statistical Bulletin: Births in England and Wales, 2013. Titchfield, United Kingdom, Office of National Statistics, 2014 5. Flenady V, Frøen JF, Pinar H, Torabi R, Saastad E, Guyon G, Russell L, Charles A, Harrison C, Chauke L, Pattinson R, Koshy R, Bahrin S, Gardener G, Day K, Petersson K, Gordon A, Gilshenan K: An evaluation of classification systems for stillbirth. BMC Pregnancy Childbirth 2009, 9:24 6. Heazell AEP, Worton SA, Higgins LE, Ingram E, Johnstone ED, Jones RL, Sibley CP: IFPA Gábor Than Award Lecture: recognition of
The American Journal of Pathology
-
20.
21.
22.
23.
24.
placental failure is key to saving babies’ lives. Placenta 2015, 36(Suppl 1):S20eS28 Ptacek I, Sebire NJ, Man JA, Brownbill P, Heazell AEP: Systematic review of placental pathology reported in association with stillbirth. Placenta 2014, 35:552e562 Redline RW: Placental inflammation. Semin Neonatol 2004, 9: 265e274 Redline RW: Villitis of unknown etiology: noninfectious chronic villitis in the placenta. Hum Pathol 2007, 38:1439e1446 Derricott H, Jones RL, Heazell AEP: Investigating the association of villitis of unknown etiology with stillbirth and fetal growth restriction a systematic review. Placenta 2013, 34:856e862 Rudzinski E, Gilroy M, Newbill C, Morgan T: Positive C4d immunostaining of placental villous syncytiotrophoblasts supports hostversus-graft rejection in villitis of unknown etiology. Pediatr Dev Pathol 2013, 16:7e13 Kim J-S, Romero R, Kim MR, Kim YM, Friel L, Espinoza J, Kim CJ: Involvement of Hofbauer cells and maternal T cells in villitis of unknown aetiology. Histopathology 2008, 52:457e464 Kim MJ, Romero R, Kim CJ, Tarca AL, Chhauy S, LaJeunesse C, Lee D-C, Draghici S, Gotsch F, Kusanovic JP, Hassan SS, Kim J-S: Villitis of unknown etiology is associated with a distinct pattern of chemokine up-regulation in the feto-maternal and placental compartments: implications for conjoint maternal allograft rejection and maternal anti-fetal graft-versus-host disease. J Immunol 2009, 182: 3919e3927 Brito H, Juliano P, Altemani C, Altemani A: Is the immunohistochemical study of the inflammatory infiltrate helpful in distinguishing villitis of unknown etiology from non-specific infection villitis? Placenta 2005, 26:839e841 Redline RW, Patterson P: Villitis of unknown etiology is associated with major infiltration of fetal tissue by maternal inflammatory cells. Am J Pathol 1993, 143:473e479 Kapur P, Rakheja D, Gomez AM, Sheffield J, Sanchez P, Rogers BB: Characterization of inflammation in syphilitic villitis and in villitis of unknown etiology. Pediatr Dev Pathol 2004, 7:453e458. discussion 421 Boog G: Chronic villitis of unknown etiology. Eur J Obstet Gynecol Reprod Biol 2008, 136:9e15 Katzman PJ, Murphy SP, Oble DA: Immunohistochemical analysis reveals an influx of regulatory T cells and focal trophoblastic STAT-1 phosphorylation in chronic villitis of unknown etiology. Pediatr Dev Pathol 2011, 14:284e293 Warrander LK, Batra G, Bernatavicius G, Greenwood SL, Dutton P, Jones RL, Sibley CP, Heazell AEP: Maternal perception of reduced fetal movements is associated with altered placental structure and function. PLoS One 2012, 7:e34851 Hamilton S, Oomomian Y, Stephen G, Shynlova O, Tower CL, Garrod A, Lye SJ, Jones RL: Macrophages infiltrate the human and rat decidua during term and preterm labor: evidence that decidual inflammation precedes labor. Biol Reprod 2012, 86:39 Girard S, Heazell AE, Derricott H, Allan SM, Sibley CP, Abrahams VM, Jones RL: Circulating cytokines and alarmins associated with placental inflammation in high-risk pregnancies. Am J Reprod Immunol 2014, 72:422e434 Bachmayer N, Rafik Hamad R, Liszka L, Bremme K, SverremarkEkström E: Aberrant uterine natural killer (NK)-cell expression and altered placental and serum levels of the NK-cell promoting cytokine interleukin-12 in pre-eclampsia. Am J Reprod Immunol 2006, 56: 292e301 de Moraes-Pinto MI, Vince GS, Flanagan BF, Hart CA, Johnson PM: Localization of IL-4 and IL-4 receptors in the human term placenta, decidua and amniochorionic membranes. Immunology 1997, 90: 87e94 Myerson D, Parkin RK, Benirschke K, Tschetter CN, Hyde SR: The pathogenesis of villitis of unknown etiology: analysis with a new conjoint immunohistochemistry-in situ hybridization procedure to
ajp.amjpathol.org
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
9
1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116
1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132Q14 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178
Derricott et al
25.
26. 27. 28.
29.
30.
31. 32.
33.
34. 35. 36.
10
identify specific maternal and fetal cells. Pediatr Dev Pathol 2006, 9: 257e265 Labarrere C, Faulk W, McIntyre J: Villitis in normal term human placentae: frequency of the lesion determined by monoclonal antibody to HLA-DR antigen. J Reprod Immunol 1989, 16:127e135 Krieger NR, Yin DP, Fathman CG: CD4þ but not CD8þ cells are essential for allorejection. J Exp Med 1996, 184:2013e2018 Chaplin DD: Overview of the immune response. J Allergy Clin Immunol 2010, 125(2 Suppl 2):S3eS23 Feeley L, Mooney EE: Villitis of unknown aetiology: correlation of recurrence with clinical outcome. J Obstet Gynaecol 2010, 30: 476e479 Ito Y, Matsuoka K, Uesato T, Sago H, Okamoto A, Nakazawa A, Hata K: Increased expression of perforin, granzyme B, and C5b-9 in villitis of unknown etiology. Placenta 2015, 36:531e537 Medawar PB: Some immunological and endocrinological problems raised by evolution of viviparity in vertebrates. Symp Soc Exp Biol 1953, 7:320e328 Challis JR, Lockwood CJ, Myatt L, Norman JE, Strauss JF 3rd, Petraglia F: Inflammation and pregnancy. Reprod Sci 2009, 16:206e215 Raghupathy R, Makhseed M, Azizieh F, Hassan N, Al-Azemi M, AlShamali E: Maternal Th1- and Th2-type reactivity to placental antigens in normal human pregnancy and unexplained recurrent spontaneous abortions. Cell Immunol 1999, 196:122e130 Cousins DJ, Lee TH, Staynov DZ: Cytokine coexpression during human Th1/Th2 cell differentiation: direct evidence for coordinated expression of Th2 cytokines. J Immunol 2002, 169:2498e2506 Perricone C, de Carolis C, Perricone R: Pregnancy and autoimmunity: a common problem. Best Pract Res Clin Rheumatol 2012, 26:47e60 Jamieson DJ, Theiler RN, Rasmussen SA: Emerging infections and pregnancy. Emerg Infect Dis 2006, 12:1638e1643 Lombardelli L, Aguerre-Girr M, Logiodice F, Kullolli O, Casart Y, Polgar B, Berrebi A, Romagnani S, Maggi E, Le Bouteiller P,
37.
38.
39.
40.
41.
42.
43. 44.
45.
Piccinni M-P: HLA-G5 induces IL-4 secretion critical for successful pregnancy through differential expression of ILT2 receptor on decidual CD4þ T cells and macrophages. J Immunol 2013, 191:3651e3662 Alijotas-Reig J, Llurba E, Gris JM: Potentiating maternal immune tolerance in pregnancy: a new challenging role for regulatory T cells. Placenta 2014, 35:241e248 Ingman K, Cookson VJ, Jones CJP, Aplin JD: Characterisation of Hofbauer cells in first and second trimester placenta: incidence, phenotype, survival in vitro and motility. Placenta 2010, 31:535e544 Burton GJ, Kaufmann P, Huppertz B: Anatomy and genesis of the placenta. Edited by Neill JD. The Physiology of Reproduction. ed 3. St. Louis, Elsevier, 2006, pp 211e266 Mullins E, Prior T, Roberts I, Kumar S: Changes in the maternal cytokine profile in pregnancies complicated by fetal growth restriction. Am J Reprod Immunol 2012, 68:1e7 Mullins E, Prior T, Roberts I, Kumar S: Changes in the fetal and neonatal cytokine profile in pregnancies complicated by fetal growth restriction. Am J Reprod Immunol 2013, 69:441e448 Young OM, Tang Z, Niven-Fairchild T, Tadesse S, Krikun G, Norwitz ER, Mor G, Abrahams VM, Guller S: Toll-like receptormediated responses by placental Hofbauer cells (HBCs): a potential pro-inflammatory role for fetal M2 macrophages. Am J Reprod Immunol 2015, 73:22e35 Altemani A, Gonzatti A, Metze K: How many paraffin blocks are necessary to detect villitis? Placenta 2003, 24:116e117 Barnes DM, Harris WH, Smith P, Millis RR, Rubens RD: Immunohistochemical determination of oestrogen receptor: comparison of different methods of assessment of staining and correlation with clinical outcome of breast cancer patients. Br J Cancer 1996, 74: 1445e1451 Khong TY: Expression of MHC class II antigens by placental villi: no relationship with villitis of unknown origin. J Clin Pathol 1995, 48: 494e495
ajp.amjpathol.org
-
The American Journal of Pathology
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240
1241 1242 1243 1244 1245 1246 1247
Supplemental Figure S1
Representative images of placental sections immunostained for cytokines. A, C, and D: VUE lesions were stained for IL-10 (A), IL-6 (C), and TGF-b (D). B: Cytokine staining of a placental section from NP immunostained for IL-10. Scale bar Z 50 mm. Original magnification, 100. NP, normal pregnancy; TGF, transforming growth factor; VUE, villitis of unknown etiology.
Q13
Supplemental Figure S2 Quantification of cytokine staining intensity using HistoQuest image analysis software. IL-2 (A), IL-12 (B), IL-4 (C), and IL-10 (D). Horizontal lines represent median. *P < 0.05, **P < 0.01, and ***P < 0.001 by Kruskal-Wallis test with Dunn’s post hoc test. DAB, diaminobenzidine; NP, normal pregnancy; SB FGR, stillbirth with fetal growth restriction; LB FGR, live birth with fetal growth restriction.
FLA 5.4.0 DTD AJPA2259_proof 3 February 2016 6:58 pm EO: AJP15_0441
1248 1249 1250 1251 1252 1253 1254