The American Journal of Pathology, Vol. -, No. -, - 2018

1 63 2 64 3 65 4 66 5 67 6 ajp.amjpathol.org 68 7 69 8 70 9 71 10 72 11 73 12 Trefoil Factor 2 Promotes Type 2 Immunity and 74 13 75 14 Lung Repair through Intrinsic Roles in 76 15 77 16 78 17 Hematopoietic and Nonhematopoietic Cells 79 18 80 Q21 19 Li-Yin Hung, Taylor K. Oniskey, Debasish Sen, Matthew F. Krummel, Andrew E. Vaughan, Noam A. Cohen, and 81 Q2 20 De’Broski R. Herbert 82 21 83 22 84 From the School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 23 85 24 86 25 Accepted for publication 87 Trefoil factors (TFFs) are small secreted proteins that regulate tissue integrity and repair at mucosal 26 January 23, 2018. 88 surfaces, particularly in the gastrointestinal tract. However, their relative contribution(s) to controlling 27 Address correspondence to 89 baseline lung function or the extent of infection-induced lung injury are unknown issues. With the use 28 De’Broski R. Herbert, Ph.D., 90 of irradiation bone marrow chimeras, we found that TFF2 produced from both hematopoietic- and 29 School of Veterinary Medicine, 91 nonhematopoieticederived cells is essential for host protection, proliferation of alveolar type 2 cells, 30 University of Pennsylvania, 92 Q5 and restoration of pulmonary gas exchange after infection with the hookworm parasite Nippostrongylus 31 Philadelphia, PA 19104.E-mail: 93 [email protected]. brasiliensis. In the absence of TFF2, lung epithelia were unable to proliferate and expressed reduced 32 94 lung mRNA transcript levels for type 2 response-inducing IL-25 and IL-33 after infectious injury. 33 95 Strikingly, even in the absence of infection or irradiation, TFF2 deficiency compromised lung structure 34 96 and function, as characterized by distended alveoli and reduced blood oxygen levels relative to wild- 35 97 type control mice. Taken together, we show a previously unappreciated role for TFF2, produced by 36 98 either hematopoietic or nonhematopoietic sources, as a pro-proliferative factor for lung epithelial cells 37 99 under steady-state and infectious injury conditions. (Am J Pathol 2018, -:1e10; https://doi.org/ 38 100 10.1016/j.ajpath.2018.01.020) 39 101 40 102 41 103 42 104 Q6 43 Coordination of host immunity and tissue repair after receptor signaling to promote bronchial epithelial cell 105 18 44 infection involves a diverse array of secreted and migration and coexpression of TFF1 and TFF2 in Clara 106 45 cell-associated molecules. Indeed, mucosal cytokine cells after acute lung injury suggests that they may contribute 107 46 production at sites of injury caused by pathogens, chemicals, to tissue regeneration.19 Of interest, a genetic screen identi- 108 47 and/or abrasions has a major impact on the healing process.1 fied TFF2 as a potential regulator of pulmonary function,20,21 109 48 TFF family proteins (TFF1 to TFF3) are reparative proteins but no evidence has found that it is important for gas 110 49 secreted at sites of injury that drive epithelial migration over exchange, tissue regeneration, or baseline lung architecture 111 50 areas of denuded basement membrane,2 promote cytoskeletal under steady-state or postinjury conditions. 112 51 rearrangement, and inhibit apoptosis.3,4 Even though trefoils Both pulmonary and intestinal epithelia produce TFF2 in 113 52 114 are well-documented regulators of gastrointestinal injury,5e7 the context of type 2 inflammation caused by allergen or 53 15 115 54 their role(s) in extraintestinal sites such as bladder, brain, hookworm infection. However, the role of TFF2 as a 116 55 intestine, oral cavity, kidney, liver, spleen, thymus, and lung driver or suppressor of allergic lung pathologic process is 117 8e12 56 are less well understood. 118 57 Lung pathologic processes induced by allergic asthma, 119 58 worm infection, smoking, tumorigenesis, chronic obstructive Supported by NIH grants R00HL131817 (A.E.V.), R01DC013588 Q3 120 59 pulmonary disease, and naphthalene-induced lung injury all (N.A.C.), R01GM083204 (D.R.H.), R01AI095289 (D.R.H.), and Q4 121 U01AI125940 (D.R.H.), McCabe Pilot Fund Award 015802 (A.E.V.), and 60 13e17 122 induce TFF2 production by epithelial cells. TFF2 and Veterans Administration Merit Review grant CX001617 (N.A.C.). 61 TFF3 can function in synergy with epidermal growth factor Disclosures: None declared. 123 62 124

Copyright ª 2018 Published by Elsevier Inc. on behalf of the American Society for Investigative Pathology. https://doi.org/10.1016/j.ajpath.2018.01.020 FLA 5.5.0 DTD Š AJPA2856_proof Š 13 March 2018 Š 3:29 pm Š EO: 2017_676 Hung et al

125 controversial.16,22 Moreover, although TFF2 expression in were irradiated with 10 Gy administered as a split dose and 187 126 epithelial cells is well known, recent evidence shows that were retro-orbitally inoculated with 2 to 5 106 total BM 188 127 TFF2 is also expressed more broadly in the hematopoietic cells isolated from the femurs of the donor animals within 1 189 128 190 compartment, including CD4þ T cells and peritoneal hour after the second dose of irradiation. All irradiated 129 191 macrophages.23 To determine whether hematopoietic- or recipients were administered 1% enrofloxacin for 2 weeks 130 fi 192 131 nonhematopoieticederived TFF2 was important for the after irradiation and were evaluated for chimerism ef ciency 193 132 production of type 2 cytokines, worm clearance, and/or at 6 to 8 weeks after transfer. Only animals that showed >90% 194 133 restoration of lung function after infection-induced tissue donor chimerism were used for subsequent data analysis. 195 134 damage, we turned to a Nippostrongylus brasiliensis 196 135 infection model, which partially recapitulates the transient Pulse Oximetry 197 136 lung damage caused by human worm infections.24 Data 198 137 show that hematopoietic-derived TFF2 is essential for Pulse oximetry of mice were measured with MouseOx Plus 199 138 hookworm-induced TFF2 production, host immunity, and with small CollarClip (both from Starr Life Sciences Corp., 200 139 the proliferation of alveolar type 2 cells after lung injury. Oakmont, PA). Hair from the sensor site on the mice was 201 140 202 Nonhematopoietic-derived TFF2 was also important for removed 1 day before oximetry as the manufacturer’s 141 203 host immunity and lung tissue repair. Unexpectedly, even 142 manual suggested. On the day of measurement, mice were 204 143 under homeostatic conditions without deliberate tissue either restrained or lightly anesthetized with ketamine/ 205 fi 144 damage, we found that loss of TFF2 signi cantly reduced xylazine, and 1 minute of error-free data were recorded and 206 145 pulmonary function compared with wild-type (WT) mice. used in analysis. No significant differences were observed in 207 146 Taken together, these data reveal an unexpected role for the data readings between these two methods. Mice were 208 147 TFF2 as an essential part of lung tissue maintenance and kept on a heating pad (37C) during oximetry to avoid 209 148 restoration after infection in mice. hypothermia. Results were presented as raw oxygen 210 149 211 saturation (SpO2), which was a readout from MouseOx 150 212 Plus, or normalized to baseline SpO2, followed by calcula- 151 Materials and Methods tion of the percentage of change from the initial reading in 213 152 214 the same animal. For hookworm infection studies, mice 153 Mice and N. brasiliensis Model 215 154 were tattooed to ensure longitudinal data collection. 216 155 All mice, including WT C57BL/6, C57BL/6 Ly5.1 (BoyJ), 217 fi 156 TFF2-de cient mice (previously backcrossed for eight gen- Vital Imaging of Lung Slices 218 157 erations to C57BL/6), and chicken actinecyan fluorescent 219 158 protein (CFP) transgenic mice have been previously For vital laser scanning confocal imaging, sections of lungs 220 159 described.25 Mice were bred in house in a fully equipped 400 mm thick were prepared as previously described.26 221 160 specific pathogen-free barrier facility, staffed, and maintained Briefly, mice were euthanized with a lethal dose of 1.3% 222 161 according to the guidelines specified by the pertinent state and tribromoethanol (Avertin; Sigma-Aldrich, St. Louis, MO) 223 162 federal regulations for vivaria at University of California at and exsanguinated. Exposed lungs were then intratracheally 224 163 San Francisco or University of Pennsylvania. All procedures inflated with 1.2 mL of 37 C 2.0% low melting temperature 225 164  226 were reviewed and approved by Institutional Animal Care agarose (Fisher Scientific, Hampton, NH), and solidified 165 227 166 and Use Committee at University of California at San with cold phosphate-buffered saline (8C to 12C). The left 228 167 Francisco (protocol AN109782-01) or the University of lobes were isolated, sliced into thick sections (400 mm) with 229 168 Pennsylvania (protocol 805911). Sex-matched naive mice a Leica Vibratome VT1000S (Wetzler, Germany), mounted 230 169 aged between 6 and 12 weeks were subcutaneously on plastic coverslips with the use of Vetbond (3M, Maple- 231 170 inoculated with 500 to 750 infective stage larvae collected wood, MN), and then imaged on a temperature-controlled 232 171 from 7-dayeold coprocultures maintained in the laboratory at stage (Warner Instruments, Hamden, CT) maintained at 233 172 25C. Parasites collected in sterile 1 phosphate-buffered 37C, while in constant perfusion with oxygenated (95% 234 173  235 saline that contained penicillin/streptomycin were washed O2; 5% CO2) RPMI without phenol red. Images were 174 several times through repeated centrifugation before inocu- acquired with a Nikon A1R microscope (Tokyo, Japan) 236 175 lation. Infected mice were euthanized by carbon dioxide equipped with an automated Prior XY stage, according to 237 176 238 narcosis at 9 days after inoculation unless specified otherwise. the manufacturer’s protocol and software, and the parame- 177 239 178 Adult worms were collected from the entire length of the ters described here. For excitation a 5-W 740- to 1000-nm 240 179 small intestine opened longitudinally spanning the duodenum tunable MaiTai HP (Newport, Santa Clara, CA) laser was 241 to proximal ileum and placed into a Baermann apparatus, and used. A 20 0.95 N.A. water-immersion objective 180  242 181 all of the adult worms collected after 2 to 3 hours were (Olympus XLUMPLFLN 20XW; Olympus, Tokyo, Japan) 243 182 counted under a stereo-microscope at 5 magnification. was used, and a spatial resolution of 1.13 mm/pixel was 244 183 Bone marrow (BM) chimeras were generated between achieved. Two micron Z-depth per plane was used for 245 184 TFF2-deficient and CD45.1 age- and sex-matched strains three-dimensional imaging. Images were recorded with the 246 185 randomly assigned as donors or recipients. The recipients galvanometer-based scanning mode. For CFP excitation, the 247 186 248

2 ajp.amjpathol.org - The American Journal of Pathology FLA 5.5.0 DTD Š AJPA2856_proof Š 13 March 2018 Š 3:29 pm Š EO: 2017_676 Hematopoietic-Derived TFF2 Promotes Lung Repair Q1

249 laser was tuned to 900 nm and a 450/50 nm emission band Enzyme-Linked Immunosorbent Assay 311 250 Q7 pass was used. Images were analyzed with Imaris (Bitplane, 312 251 Concord, MA) and Matlab (Math Works, Natick, MA). For Mouse lungs were lavaged with 1 mL of phosphate-buffered 313 314 252 evaluating alveolar cross-sectional area, individual sections saline during necropsy. After centrifugation at 1500 rpm for 253 3 315 of 200 200 50 m from the three-dimensional imaging 5 minutes the supernatants were stored at 80C before 254 m 316   enzyme-linked immunosorbent assay. MouseÀ Trefoil Factor 255 volume were selected, and alveolar area was calculated as 317 256 the area of contiguous dark space or the absence of CFP 2 ELISA (enzyme-linked immunosorbent assay) kit was 318 257 with the use of Matlab scripts for each plane in the purchased from United States Biological (Swampscott, 319 258 three-dimensional volume by using a cutoff 2000 mm2. MA). 320 259  321 260 Quantitative Real-Time PCR Statistical Analysis 322 261 323 262 Total RNA was harvested from lung or intestinal tissues with Sample sizes were not determined statistically before 324 263 the use of RNeasy Mini kit (Qiagen, Hilden, Germany) experiments. All mice were randomly assigned to experi- 325 264 326 according to the manufacturer’s protocol. Total RNA (500 ng) mental or control groups. All data shown are means and 265 327 SEM from four to six mice per group unless otherwise 266 was reverse-transcribed with Superscript II (Invitrogen, 328 267 Carlsbad, CA) according to the manufacturer’s protocol. One indicated. Experiments were repeated at least twice with 329 268 to four diluted cDNA samples were added to SsoAdavanced representative data shown. Grubb’s test was performed for 330 269 SYBR Green Supermix (Bio-Rad, Hercules, CA), and real- each data set to exclude outliers. For comparison unpaired 331 270 time PCR reactions were run on CFX96 Real-Time PCR t-test was used to compare two groups and one-way analysis 332 271 detection system (Bio-Rad). Gene expression is normalized to of variance was used to compare three or more groups. 333 272 Gapdh, and data are presented as means SEM from the Two-way analysis of variance was used to compare data 334 273 replicates. Primers used in this study includedÆ the following: from repeated measures of same animals for longitudinal 335 274 336 Gapdh forward: 50-AGGTCGGTGTGAACGGATTTG-30, analysis. Statistics were performed with GraphPad Prism 275 and reverse: 5 -TGTAGACCATGTAGTTGAGGTCA-3 ; version 6 (GraphPad Software, Inc., La Jolla, CA). In all 337 276 0 0 338 Il25 forward: 5 -GCTGTTGCTGAAGAAGGTAGT-3 , and cases P < 0.05 was considered statistically significant. 277 0 0 339 278 reverse: 50-TTCAAGTCCCTGTCCAAC-30; Il33 forward: 50- 340 279 TCCCAACAGAAGACCAAAG-30, and reverse: 50-GATA- Results 341 280 CTGCCAAGCAAGGAT-30; Retnla forward: 50-GGATGC- 342 281 CAACTTTGAATAGG-30, and reverse: 50-GCACACCC- Hematopoietic CelleDerived TFF2 Serves an Important 343 282 AGTAGCAGTC-30); Retnlb forward: 50-GATACTGC- Role in Host Defense after Infection with the 344 283 CAAGCAAGGAT-30, and reverse: 50-AACACAGTGTAG- Hookworm Parasite N. brasiliensis 345 284 GCTTCATGCTGTA-30; Spc forward: 50-AGCAAAGAGG- 346 285 347 TCCTGATGGA-30, and reverse: 50-ATGAGAAGGCGTTT- Previous demonstration that TFF2 deficiency caused 286 348 GAGGTG-30; and Ifng forward: 50-GTGGCATAGATGT- myeloid cytokine dysregulation, combined with evidence 287 15,23 349 GGAAGAA-3 , and reverse: 5 -GCTGTTGCTGAA- that TFF2 can be expressed in CD4þ T cells, prompted 288 0 0 350 GAAGGTAGT-3 . us to interrogate whether hematopoietically derived TFF2 289 0 351 290 plays a critical role in the immune response and/or resolu- 352 291 Flow Cytometry tion of lung pathologic processes after N. brasiliensis 353 292 infection. To address this question, irradiation BM chimeras 354 / 293 The following antibodies were purchased from BioLegend were generated between WT (CD45.1) and Tff2À À 355 294 (San Diego, CA): rat anti-mouse CD31 (clone 390), rat (CD45.2) strains [WT/WT capable of producing both 356 295 anti-mouse CD45 (30-F11), rat anti-mouse CD49f/a6 integrin hematopoietically and nonhematopoietically derived TFF2, 357 296 (GoH3), rat anti-mouse CD104/b4 integrin (346-11A), rat knockout (KO)/WT only capable of producing non- 358 297 anti-mouse F4/80 (BM8), and rat anti-mouse CD326/epithelial hematopoietically derived TFF2, WT/KO only capable of 359 298 Q8 cell adhesion molecule (EpCAM; G8.8). Hamster anti-mouse producing hematopoietically derived TFF2, and KO/KO 360 299 Podoplanin/T1a (eBio8.1.1), rat anti-mouse CD11b (M1/70), unable to produce TFF2]. Six weeks after lethal whole-body 361 300 362 hamster anti-mouse CD11c (N418), rat anti-mouse I-A/I-E irradiation (10 Gy) and BM reconstitution (Figure 1A), F1 301 fi ½ Š 363 302 (M5/114.15.2), hamster anti-mouse CD103 (2E7), and rat anti- >95% donor chimerism was con rmed in peripheral CD4þ, 364 303 mouse Sca-1 (D7) were purchased from eBioscience (San B220þ, and CD11bþ populations (Figure 1,BeD) as well 365 304 Diego, CA). Cell viability was determined by LIVE/DEAD as alveolar macrophages defined as CD45þ, CD11cþ, 366 305 Fixable Aqua Stain (Life Technologies, Carlsbad, CA) and CD64þ, and Siglec-Fþ by flow cytometry with the use of 367 306 Annexin V (BD Biosciences, San Jose, CA). Rat anti-mouse monoclonal antibody specific for the congenic markers 368 / 307 Siglec-F (E50-2440) was purchased from BD Biosciences. CD45.1 (WT) and CD45.2 (TFF2À À )(Figure 1,EeG) in 369 308 Flow cytometry was performed on LSRII machine (BD all combinations tested. Therefore, TFF2 deficiency did not 370 309 Q9 Biosciences) with BD FACSDiva software. have a negative impact on BM engraftment. 371 310 372

The American Journal of Pathology - ajp.amjpathol.org 3 FLA 5.5.0 DTD Š AJPA2856_proof Š 13 March 2018 Š 3:29 pm Š EO: 2017_676 Hung et al

373 435 374 436 375 437 376 438 377 439 378 440 379 441 380 442 381 443 382 444 383 445 384 446 385 447 386 448 387 449 388 450 389 451 390 452 391 453 392 454 393 455 394 456 395 Figure 1 Generation and confirmation of bone marrow (BM) chimera from wild-type (WT) Q15457 / 396 and trefoil factor (TFF)2À À mice. A: Schematic describing irradiation and the generation of BM 458 / 397 chimera between WT C57BL/6 (CD45.1) and Tff2À À (CD45.2) mice. Mice were lethally irradi- 459 398 ated, intravenously reconstituted with total BM cells from the opposite strain, rested for 6 weeks 460 399 for reconstitution, followed by s.c. inoculation with 750 Nippostrongylus brasiliensis infectious 461 e 400 larvae L3. B D: Chimerism in peripheral blood cells 6 weeks after initial BM transfer. Expression 462 of congenital marker CD45.1 versus CD45.2 on CD4þ (B), CD11bþ (C), and B220þ (D) cells in the 401 463 blood. E: Flow gating strategy to identify alveolar macrophages (aMac) from total lung digests Q16 402 464 that are singlet, live, and CD45þ. F and G: Chimerism of aMac in naive (F) and day 4 after N. 403 brasiliensis infection (G). KO, knockout; N.b., N. brasiliensis. Q17465 404 466 405 467 406 468 407 469 408 To test for the relative importance of hematopoietically with WT/WT chimeras (Figure 2E). Conversely, lung 470 409 versus nonhematopoietically derived TFF2 in gastrointes- transcripts for Ifng, the signature gene of type 1 responses, 471 410 tinal host defense, the chimeric mice were inoculated with were most highly expressed in KO/KO chimeras, followed 472 411 the infectious stage larvae of the hookworm parasite N. by KO/WT chimeras, and the lowest levels were observed 473 412 474 brasiliensis. WT/WT chimeras were significantly better in the WT/KO and WT/WT groups (Figure 2F). Thus, 413 475 414 protected than any other chimeric combinations at day 9 these data suggested that both hematopoietically and 476 415 after infection, as evidenced by reduced worm burden nonhematopoietically derived TFF2 regulate worm expulsion 477 F2 (Figure 2A). Congruent with a host protective response, it and substantially affect the balance between type 1 and type 2 416 ½ Š 478 417 was found that resistin-like molecule (RELM)b intestinal responses during hookworm infection. 479 418 mRNA transcripts were twofold to threefold higher in the 480 419 WT/WT chimeras than in chimeric mice lacking hema- TFF2 from Either Hematopoietic or Nonhematopoietic 481 420 topoietically derived TFF2 (KO/WT), nonhematopoieti- Sources Protects against Hookworm-Induced Lung 482 421 cally derived TFF2 (WT/KO), or both (KO/KO) Pathologic Processes 483 422 (Figure 2B). 484 423 TFF2 deficiency impaired IL33 mRNA induction in Next, it was tested whether TFF2 was required for repair of 485 424 486 myeloid antigen-presenting cells, suggesting that TFF2 may damaged lung tissue, and, if so, whether hematopoietic or 425 15 487 426 be an important modulator of type 2 responses. To further nonhematopoietic sources were important. Lung damage in the 488 427 investigate whether type 2 cytokine expression was impaired infected cohorts of WT/WT, KO/WT, WT/KO, and 489 428 by TFF2 deficiency in hematopoietic or nonhematopoietic KO/KO chimeric mice infected as described in Mice and N. 490 429 cells, lung mRNA transcripts were evaluated at day 9 by brasiliensis Model was evaluated by pulse oximetry and his- Q11 491 430 Q10 quantitative real-time RT-PCR. Transcripts for Il33 and Il25 tologic assessment of formalin-fixed, paraffin-embeddedein- 492 431 were uniformly reduced in TFF2-deficient chimeras flated lungs. Data show a similar SpO2 nadir at day 3 among 493 432 (Figure 2, C and D), whereas Retnla transcripts were twofold strains, reflective of the peak of hookworm-induced lung 494 433 495 lower only in KO/KO and KO/WT chimeras compared injury. However, the rebound in SpO2 levels at day 9 that was 434 496

4 ajp.amjpathol.org - The American Journal of Pathology FLA 5.5.0 DTD Š AJPA2856_proof Š 13 March 2018 Š 3:29 pm Š EO: 2017_676 Hematopoietic-Derived TFF2 Promotes Lung Repair

497 559 498 560 499 561 500 562 501 563 502 564 503 565 504 566 505 567 506 568 507 569 508 570 509 571 510 572 511 573 512 574 513 575 514 576 515 577 516 578 517 579 518 580 519 581 520 582 521 583 Figure 2 Defective worm expulsion, gastrointestinal resistin-like molecule b expression, and innate type 2 cytokine production in Nippostrongylus bra- 522 584 siliensiseinfected chimeras lacking hematopoietically or nonhematopoietically derived trefoil factor 2. A and B: Numbers of N. brasiliensis adult worms 523 585 recovered from the intestinal lumen (A) and Retnlb mRNA transcript levels within the jejunum of irradiation bone marrow chimeras at 9 days after infection 524 (B). CeF: Quantitative lung mRNA expression levels of Il33 (C), Il25 (D), Retnla (E), and Ifng (F) on day 9 after N. brasiliensis infection. Data are expressed as 586 525 means SEM with each symbol representing one mouse. *P < 0.05, **P < 0.01 (one-way analysis of variance). KO, knockout; WT, wild-type. 587 526 Æ 588 527 589 528 observed in the WT/WT chimeras was significantly blunted distal lung epithelia were evaluated with flow cytometry to 590 529 in the KO/WT and KO/KO chimeras and partially calculate the percentage of BrdUþ cells. WT/WT chimeras 591 530 592 Q12 F3 impaired in the WT/KO chimeras (Figure 3, B and C). The had higher percentages of EpCAMþBrdUþ cells that were also 531 ½ Š 593 histologic features of N. brasiliensiseinfected lung tissues pro-SpCþ [surfactant-associated protein C; alveolar type 2 Q14 532 594 corroborated the persistent defects in gas exchange, with (ATII) cells] compared with chimeras lacking TFF2 in either 533 595 534 equivalent injury at day 3 (not shown), but persistent areas of the hematopoietic or nonhematopoietic compartment 596 denuded distal lung space lacking alveoli within both (Figure 4, A and B). In addition, the total number of EpCAMþ F4 535 ½ Š 597 536 KO/WT and KO/KO chimeras (Figure 3D). Quantifica- cells recovered from the distal lung compartment was higher in 598 537 tion of lung damage by using mean linear intercept revealed a the WT/WT chimeras than in all other TFF2-deficient chi- 599 538 significant reduction in KO/WT and KO/KO chimeras meras (Figure 4C). Real-time PCR analysis was used to 600 539 compared with the WT/WT group (Figure 3E). Intriguingly, confirm higher lung mRNA transcripts for Spc (lineage marker 601 540 significant differences in the number or quality of inflamma- for ATII cells) in WT/WT lung compared with expression 602 541 tory cell infiltration in bronchoalveolar lavage fluid among levels in WT/KO and KO/KO chimeras (Figure 4D). 603 542 chimeras and WT mice were not observed, although KO mice These data implied an essential role for TFF2 for maintaining 604 543 had reduced numbers of eosinophils and lymphocytes the ATII cell population in the lung after hookworm injury. 605 544 606 (Figure 3F). These data indicated a necessary role for both 545 607 hematopoietically and nonhematopoietically derived TFF2 in 546 TFF2 Deficiency Leads to Reduced Gas Exchange and 608 547 the repair of lung tissue damaged by hookworm larvae. 609 548 Abnormal Alveolar Architecture during Homeostasis 610 549 611 550 TFF2 Is Important for Epithelial Replenishment after KO/KO chimeras consistently fared worse than all other 612 551 Lung Injury Caused by N. brasiliensis Infection chimeras after hookworm infection. Even under baseline 613 552 conditions, the SpO2 levels in KO/KO chimeras were 614 553 It was next determined whether the impaired SpO2 rebound moderately lower than that of the other chimeras (Figure 5A). F5 615 ½ Š 554 and increased lung damage observed in TFF2-deficient mice Given that quantitative trait loci mapping studies in mice 616 555 were associated with defects in epithelial cell turnover. To identified TFF2 as one of several genes linked to baseline lung 617 556 identify S-phase lung epithelia, mice were administered function,20,21 it was tested whether TFF2 deficiency, even in 618 557 Q13 bromodeoxyuridine (BrdU) 24 hours before euthanasia, and the absence of infectious injury, compromised lung function, 619 558 620

The American Journal of Pathology - ajp.amjpathol.org 5 FLA 5.5.0 DTD Š AJPA2856_proof Š 13 March 2018 Š 3:29 pm Š EO: 2017_676 Hung et al

621 683 622 684 623 685 624 686 625 687 626 688 627 689 628 690 629 Figure 3 Both hematopoietic and non- 691 630 hematopoietic sources contribute to trefoil factor 692 631 (TFF)2 production, raw oxygen saturation (SpO2) 693 632 changes, and pathologic processes after Nippos- 694 633 trongylus brasiliensis infection. A: TFF2 levels in 695 634 bronchoalveolar (BAL) fluid harvested from mice at 696 635 indicated time points after N. brasiliensis infec- 697 tion. and Percentage of change ( SpO ) 636 B C: D 2 698 caused by infection at 3 (B) or 9 (C) days after 637 699 infection. D: Stitched images of lung tissues from 638 chimeric mice at day 9 after N. brasiliensis infec- 700 639 tion. The arrowheads indicate areas of denuded 701 640 distal lung space. E: Lung images (day 9) were 702 641 taken and stitched as in A and were analyzed with 703 642 AGI script. F: Differential counts of cells recovered 704 643 from BAL on day 9 after N. brasiliensis infection. 705 Data are expressed as means SEM with each 644 Æ 706 645 symbol representing one mouse. n Z 8 to 10 mice 707 per group ( ); n Z 4 mice per group ( ); n Z 4 to 646 A E 708 6 mice per group (F); n Z 4 independent experi- 647 709 ments (F). *P < 0.05, **P < 0.01 (analysis of 648 variance). Scale bars Z 0.8 mm. Original magni- 710 649 fication, 20. Eos, eosinophil; KO, knockout; 711  650 Lymph, lymphocyte; Mac, macrophage; Neut, Q18 712 651 neutrophil; WT, wild-type. 713 652 714 653 715 654 716 655 717 656 718 657 719 658 720 659 721 660 722 661 723 662 724 663 and/or architecture. As expected, comparison of naive WT was found in the number of distal lung epithelia recovered 725 / fi 664 and TFF2À À hematoxylin and eosinestained lung tissues from TFF2-de cient mice compared with WT mice. 726 / 665 revealed that TFF2À À alveoli were larger than WT alveoli Although no increase was observed in the proportion of 727 666 (Figure 5B). To more easily visualize alveolar structure by apoptotic cells in the CD45À EpCAMþ population (data not 728 667 confocal microscopy, Tff2-deficient mice were intercrossed shown), EPCAMþ cells had lower expression of a6 b4 729 25 668 with CFP transgenic mice to facilitate quantification of lung integrin (Figure 6, A and B), a component of the F6 730 ½ Š 669 architecture by quantitative assessment of pixel intensity. hemidesmosome that tethers epithelia to basement membrane 731 / 670 Actin-CFP transgenic (WT) and TFF2À À actin-CFP trans- (Figure 6,CeF). Taken together, these data indicated that 732 671 genic mice were evaluated at 6 to 10 weeks of age, and total TFF2 promotes baseline lung function, promoted epithelial 733 672 CFP signal was used to quantitate alveolar cross-sectional proliferation, and aided epithelial attachment in the 734 673 735 area within equal distal lung volume (200 200 50 pulmonary tract. 674 3  fi  736 675 mm ). Notably, this analysis revealed that TFF2 de ciency 737 676 caused a striking increase in the frequency of large diameter 738 677 alveoli compared with WT alveoli (Figure 5C). Measurement Discussion 739 678 of SpO2 levels corroborated this architectural defect, as 740 679 shown by significantly reduced baseline levels in TFF2- TFF proteins are among the secreted factors that regulate 741 680 deficient mice compared with WT mice under steady-state tissue integrity and repair at mucosal surfaces, particularly 742 681 conditions (Figure 5D). Interestingly, a significant reduction in the gastrointestinal mucosa. Here, we demonstrate the 743 682 744

6 ajp.amjpathol.org - The American Journal of Pathology FLA 5.5.0 DTD Š AJPA2856_proof Š 13 March 2018 Š 3:29 pm Š EO: 2017_676 Hematopoietic-Derived TFF2 Promotes Lung Repair

745 807 746 808 747 809 748 Figure 4 Decreased proliferative progenitors, 810 749 total epithelial cell numbers, and SpC expression in 811 750 chimeric lungs after Nippostrongylus brasiliensis 812 infection. A and B: Percentages of total numbers 751 813 of the CD45À , EpCAMþ, SpCþ, and BrdUþ (A) and 752 814 CD45À , EpCAMþ, SpCþ, T1aþ, and BrdUþ (B) cell 753 population within distal lung digests of chimeric 815 754 mice at day 5 after N. brasiliensis infection. C: 816 755 Total number of epithelia (CD45À EpCAMþ) cells 817 756 recovered on day 5. D: Quantitative SpC expression 818 757 in lung tissues of chimeric mice on day 9 after N. 819 758 brasiliensis infection. Data are expressed as 820 759 means SEM each symbol representing one 821 Æ Z Z 760 mouse. n 6 to 8 mice per group; n 4 inde- 822 pendent experiments.*P < 0.05, **P < 0.01, and Q19 761 823 ***P < 001 determined by analysis of variance. 762 BrdU, bromodeoxyuridine; EpCAM, epithelial cell 824 763 adhesion molecule; KO, knockout; SpC, surfactant- 825 764 associated protein C; WT, wild-type. 826 765 827 766 828 767 829 768 830 769 831 770 importance of TFF2 as a regulator of distal lung architecture reduction in baseline blood oxygen content. Taken together, 832 771 and gas exchange function. Loss of TFF2 from either these data implicate TFF2 as an important regulator of 833 772 834 hematopoietic or nonhematopoietic cells caused a reduction pulmonary repair that can be derived from hematopoietic 773 835 774 in type 2 cytokines (Il25, Il33), promoted Ifng expression, sources and that is important for maintaining mucosal 836 775 and abrogated epithelial cell proliferation after hookworm homeostasis. 837 776 infection. Loss of TFF2 from all cells resulted in a marked With the use of models of N. brasiliensis and house dust 838 777 disruption of the distal lung architecture that somewhat miteeinduced type 2 responses, we previously demon- 839 778 resembled emphysematous bullae, marked by a significant strated that TFF2-deficient mice have an early defect in the 840 779 841 780 842 781 843 782 844 783 Figure 5 Trefoil factor 2 (TFF2) deficiency re- 845 784 sults in abnormally enlarged alveoli and hypoxia at 846 785 the steady state. A: Measurement of baseline 847 786 blood oxygen levels (SpO2) in chimeras at 6 to 8 848 787 weeks after bone marrow reconstitution. B: 849 Representative photomicrographs of airway and 788 850 distal lung space within naive wild-type (WT) and 789 / 851 TFF2À À mice at 6 to 8 weeks of age. Light mi- 790 croscopy images show paraffin-embedded hema- 852 791 toxylin and eosinestained lung tissue (left) and 853 792 laser-scanning confocal micrograph showing z- 854 793 projection of gray-scale images from distal lung 855 794 tissues (50-mm thick) of WT actinecyan fluo- 856 Â 795 rescent protein (CFP) transgenic mice versus Tff2 857 / 796 À À actin-CFP transgenic naive mice (right). 858 Â fi 797 Arrowheads indicate alveolar space. C: Quanti - 859 cation of alveolar cross-sectional area in B. Each 798 860 point represents area of individual alveoli. D: 799 / 861 Measurement of baseline SpO2 in WT or Tff2À À 800 mice at 6 to 8 weeks of age. Each symbol repre- 862 801 sents 1 mouse (A and D). n Z 3 independent 863 802 experiments (C). *P < 0.05 (one-way analysis of 864 803 variance); P < 0.01, P < 001 (unpaired t- 865 yy yyy 804 test). Scale bars Z 50 mm. Original magnification, 866 40. BV, blood vessel; KO, knockout. 805 Â 867 806 868

The American Journal of Pathology - ajp.amjpathol.org 7 FLA 5.5.0 DTD Š AJPA2856_proof Š 13 March 2018 Š 3:29 pm Š EO: 2017_676 930 929 928 927 926 925 924 923 922 921 920 919 918 917 916 915 914 913 912 911 910 909 908 907 906 905 904 903 902 901 900 899 898 897 896 895 894 893 892 891 890 889 888 887 886 885 884 883 882 881 880 879 878 877 876 875 874 873 872 871 870 869

print & web 4C=FPO Tff2 n aeI.Teaayi n aigwr efre ihFoJ eso .. TeSa n. sln,OR). Ashland, Inc., (TreeStar 9.9.6 version FlowJo with performed were gating and analysis The II. Gate and I T1 ubr ( numbers iue6 Figure al et Hung 8 aigsrtg o h identi the for strategy gating a À À EpCAM / À ie aaaeepesda means as expressed are Data mice. E þ and aeI,T1 II, Gate ; rfi atr2(F2 de (TFF2) 2 factor Trefoil F fCD45 of ) a À þ EpCAM , EpCAM fi aino lelrtp AI el coexpressing cells (ATI) I type alveolar of cation þ þ The . T1 , fi inyrslsi eet neihla elahso oeue ECM)a h taystate. steady the at (EpCAMs) molecules adhesion cell epithelial in defects in results ciency a lelines blue À , L .. DTD 5.5.0 FLA Æ a 6 E ihec yblrpeetn n mouse. one representing symbol each with SEM þ b 4 þ in ( A C and and E B n CD45 and ) Š niaeqarnssprtdo h ai fteepeso fintegrins of expression the of basis the on separated quadrants indicate AJPA2856_proof À EpCAM , a 6/ þ T1 , b Š nern nwl-ye(T ( (WT) wild-type in integrins 4 ajp.amjpathol.org a 3Mrh2018 March 13 þ , a 6 þ b n 4 Z þ ( D o8mice 8 to 6 and Š F :9pm 3:29 ouain ihndsa ug fnieW and WT naive of lungs distal within populations ) - h mrcnJunlo Pathology of Journal American The e group. per C A e n Tff2 and ) F: Š ecnae( Percentage O 2017_676 EO: À / À ( B A C ie aeIindicates I Gate mice. ) a and and and 6 B: D n oa cell total and ) b Representative nbt Gate both in 4 Q20 992 991 990 989 988 987 986 985 984 983 982 981 980 979 978 977 976 975 974 973 972 971 970 969 968 967 966 965 964 963 962 961 960 959 958 957 956 955 954 953 952 951 950 949 948 947 946 945 944 943 942 941 940 939 938 937 936 935 934 933 932 931 Hematopoietic-Derived TFF2 Promotes Lung Repair

993 production of IL-33 and as a result show impaired given that TFF2-mediated epithelial migration partially re- 1055 994 IL-13edominated inflammatory responses.1 IL-33, a quires an intact epidermal growth factor receptor. Future 1056 995 prototypical alarmin cytokine, is thought to be primarily studies will need to discern whether TFF2 is an important 1057 996 1058 produced by epithelial cells after tissue damage.2 In addition product of tissue macrophages, innate lymphoid cells, and/ 997 14e16 1059 to epithelial cells, macrophages and inflammatory dendritic or Foxp3þ T regulatory cells. 998 fi fi 1060 cells isolated from TFF2-de cient mice have compromised In the rst 3 days of infection, hookworm larvae cause 1061 999 1 1000 IL-33 expression. Myeloid-derived IL-33 has been reported hemorrhagic injury to lung parenchyma because of larval 1062 3,4 1001 by several other groups in the setting of allergic disease. migratory behavior and their secreted proteolytic enzymes. 1063 1002 Herein, we show by using irradiation BM chimeras that Loss of either BM- or non-BMederived TFF2 exacerbated 1064 1003 both Il25 and Il33 expression levels were reduced in lung lung pathologic processes and impaired the proliferative 1065 1004 myeloid cells from mice lacking BM-derived TFF2. In expansion of lung epithelia during the repair phase. Type II 1066 1005 addition, there was a selective reduction in Retlna, which is alveolar epithelial cells, which coexpress EpCAM and sur- 1067 1006 characteristic of M2/alternatively activated macrophages, in factant protein C, function as progenitors in the distal lung 1068 1007 mice lacking BM-derived TFF2. Together, this suggests that compartment.17 Loss of either BM- or non-BMederived 1069 1008 1070 paracrine or autocrine signaling of TFF2 in hematopoietic TFF2 reduced both the number and percentage of ATII cells 1009 1071 cells favors type 2 responses. Congruent with this inter- in S phase at day 9 after infection. This time point is 6 days 1010 1072 1011 pretation, expression levels for Ifng, the prototypical type 1 after worm egress from the lung and likely corresponds to a 1073 fl 1012 in ammatory cytokine, were highest in the lungs of mice period of peak lung healing, based on the high levels of type 1074 18 1013 lacking TFF2 in all cells and were also significantly elevated 2 cytokine production at this time. Moreover, at 9 days 1075 1014 in mice lacking either BM- or non-BMederived TFF2 after infection, WT mice completely recover their initial 1076 1015 compared with WT chimeras. Our data are consistent with a blood oxygen levels and have threefold to fourfold greater 1077 1016 demonstration that Il33 expression was significantly reduced numbers of S-phase ATII distal lung epithelia than all other 1078 1017 in the gastrointestinal tract of TFF2-deficient animals during chimeric strains, consistent with increased Spc expression 1079 1018 dextran sulfateeinduced colitis5 and previous reports of levels in WT/WT chimeras relative to KO/WT or 1080 1019 TFF2-deficient animals producing enhanced proin- KO/KO chimeras. Scott and colleagues19,20 and Marsland 1081 1020 1082 flammatory cytokines (Ifng and Il1b).6,7 et al21 demonstrated that the architectural defects caused by 1021 1083 Data suggest that TFF2 promotes type 2 macrophage hookworm infection last for 1 year and that local lung M2 1022 > 1084 1023 responses and reduces type 1 responses. Although it has been cell activation persists during this time, despite the 1085 fi 1024 shown that TFF2 de ciency leads to an enhanced M1/classi- continued emphysematous changes. However, none of these 1086 8 1025 cally activated macrophage phenoptype, rTFF2 treatment that studies evaluated the percentage or number of proliferating 1087 1026 directs macrophages to adopt an M2 phenotype in vitro could epithelia or blood oxygen content before or after hookworm 1088 1027 not be demonstrated (data not shown). Such results suggest that infection.21 From our findings, we surmise that TFF2 drives 1089 1028 TFF2 effects on macrophages may be indirect. Because a local epithelial proliferation to rapidly restore the lung 1090 1029 receptor for TFF2 has not yet been identified, determining the function and blood oxygen levels despite ongoing archi- 1091 1030 cellular targets of TFF2 signaling remains challenging. It has tectural defects. The possibility that the dysregulation of 1092 1031 been reported that CXCR4 may function as a low-affinity inflammation in the absence of TFF2 also contributed to the 1093 1032 1094 receptor for TFF2 in epithelia and lymphocytes,9 but it was reduced SpO levels and that this reduction was not solely 1033 2 1095 found that myeloid-specific CXCR4-deficient mice (LysM- due to architectural defects from reduced epithelial prolif- 1034 Cre flox/flox 1096 1035 CXCR4 ) had no defects in type 2 immunity or worm eration cannot be ruled out. 1097 1036 expulsion during N. brasiliensis infection (data not shown). Despite the widespread notion that M2 cells promote 1098 1037 Further functional studies will be greatly aided by definitive wound healing, few studies have demonstrated that re- 1099 1038 identification of the TFF2 receptor. epithelialization is macrophage dependent.22 One recent 1100 1039 The hematopoietic cell(s) sources of TFF2 were not study demonstrated that co-culture of ATII cells and BM- 1101 1040 formally identified in this study, but could be of myeloid derived macrophage yielded more colony-forming units 1102 23 1041 lineage (macrophage) or lymphoid lineage (CD4þ T than when ATII cells are cultured alone. Our work pro- 1103 1042 cells).7,10 Tissue macrophages, which are widely accepted vides a potential mechanism to explain how that could 1104 1043 as central players in wound healing/tissue repair processes, occur, in as much as secreted macrophages (or other he- 1105 1044 express TFF2 transcripts, but the functional importance of matopoietic cells) may have an important role in promoting 1106 1045 1107 myeloid-derived TFF2 awaits further study. Recently, lung repair.24 Given that alveolar macrophages are in close 1046 10 1108 1047 Dubeyovskova et al demonstrated that mature memory- contact with ATII epithelial cells in vivo, we speculate that 1109 1048 like CD4þ T cells could produce TFF2, which is not alveolar macrophages facilitate epithelial proliferation and 1110 1049 entirely without precedent, given that T regulatory cells and tissue repair through a TFF2-dependent mechanism. 1111 1050 T helper 2 effector cells can produce amphiregulin, an Interestingly, even in the absence of infectious injury or 1112 1051 epidermal growth factor receptor agonist and pro-repair irradiation, TFF2 deficiency in mice led to a striking reduc- 1113 1052 molecule.11e13 We speculate that hematopoietic tion in cell number within the distal lung compartment. This 1114 1053 cellederived TFF2 may function similar to amphiregulin, reduced cell number was linked to reduced numbers of 1115 1054 1116

The American Journal of Pathology - ajp.amjpathol.org 9 FLA 5.5.0 DTD Š AJPA2856_proof Š 13 March 2018 Š 3:29 pm Š EO: 2017_676 Hung et al

1117 epithelial cells expressing a6/b4 integrin, an essential 7. Kurt-Jones EA, Cao L, Sandor F, Rogers AB, Whary MT, Nambiar PR, 1184 1118 component of the hemidesmosome that tethers epithelia to the Cerny A, Bowen G, Yan J, Takaishi S, Chi AL, Reed G, Houghton J, 1185 1119 1186 basement membrane. The hemidesmosome facilitates Fox JG, Wang TC: Trefoil family factor 2 is expressed in murine gastric 1120 and immune cells and controls both gastrointestinal inflammation and 1187 1121 epithelial cell attachment to basement membrane through systemic immune responses. Infect Immun 2007, 75:471e480 1188 1122 binding laminin, which, in turn, facilitates a cascade of 8. Giraud AS, Pereira PM, Thim L, Parker LM, Judd LM: TFF-2 inhibits 1189 1123 intracellular events that promote survival, growth, and dif- iNOS/NO in monocytes, and nitrated protein in healing colon after 1190 1124 ferentiation.25 Indeed, this signaling cascade may partially colitis. Peptides 2004, 25:803e809 1191 1125 1192 underlie the association of TFF2 with tumor cell metastasis.26 9. Dubeykovskaya Z, Dubeykovskiy A, Solal-Cohen J, Wang TC: 1126 Secreted trefoil factor 2 activates the CXCR4 receptor in epithelial and 1193 1127 lymphocytic cancer cell lines. J Biol Chem 2009, 284:3650e3662 1194 1128 10. Dubeykovskaya Z, Si Y, Chen X, Worthley DL, Renz BW, Urbanska AM, 1195 1129 Conclusions Hayakawa Y, Xu T, Westphalen CB, Dubeykovskiy A, Chen D, 1196 1130 Friedman RA, Asfaha S, Nagar K, Tailor Y, Muthupalani S, Fox JG, 1197 1131 Our work demonstrates a previously unappreciated and Kitajewski J, Wang TC: Neural innervation stimulates splenic TFF2 to 1198 1132 critical role for hematopoietic TFF2 in the development of arrest myeloid cell expansion and cancer. Nat Commun 2016, 7:10517 1199 1133 type 2 immune responses, host protection, and ATII pro- 11. Zaiss DM, van Loosdregt J, Gorlani A, Bekker CP, Grone A, Sibilia M, 1200 1134 1201 liferation. In addition, our demonstration that TFF2 defi- van Bergen en Henegouwen PM, Roovers RC, Coffer PJ, Sijts AJ: 1135 Amphiregulin enhances regulatory T cell-suppressive function via the 1202 1136 ciency leads to lung function defects in the absence of epidermal growth factor receptor. Immunity 2013, 38:275e284 1203 1137 deliberate injury implies a broader role for TFF2 than pre- 12. Zaiss DM, Yang L, Shah PR, Kobie JJ, Urban JF, Mosmann TR: 1204 1138 viously thought. Together, these findings further emphasize Amphiregulin, a TH2 cytokine enhancing resistance to nematodes. 1205 1139 the importance of identifying and validating molecular Science 2006, 314:1746 1206 1140 1207 pathways responsible for the downstream effects of this 13. Arpaia N, Green JA, Moltedo B, Arvey A, Hemmers S, Yuan S, 1141 Treuting PM, Rudensky AY: A distinct function of regulatory T cells 1208 1142 important mucosal cytokine. in tissue protection. Cell 2015, 162:1078e1089 1209 1143 14. Monticelli LA, Osborne LC, Noti M, Tran SV, Zaiss DM, Artis D: IL- 1210 1144 33 promotes an innate immune pathway of intestinal tissue protection 1211 1145 Acknowledgments dependent on amphiregulin-EGFR interactions. Proc Natl Acad Sci U 1212 1146 S A 2015, 112:10762e10767 1213 1147 L.-Y.H. and D.R.H. designed the experiments and wrote the 15. Burzyn D, Kuswanto W, Kolodin D, Shadrach JL, Cerletti M, Jang Y, 1214 1148 manuscript; L.-Y.H., T.K.O., and D.S. performed experi- Sefik E, Tan TG, Wagers AJ, Benoist C, Mathis D: A special population of 1215 1149 regulatory T cells potentiates muscle repair. Cell 2013, 155:1282e1295 1216 1150 ments and analyzed data; A.E.V. and N.A.C. cowrote the 1217 fl 16. Meng C, Liu G, Mu H, Zhou M, Zhang S, Xu Y: Amphiregulin may be 1151 manuscript; M.F.K. provided actinecyan uorescent pro- a new biomarker of classically activated macrophages. Biochem Bio- 1218 1152 tein mice and assisted with confocal imaging. phys Res Commun 2015, 466:393e399 1219 1153 17. Barkauskas CE, Cronce MJ, Rackley CR, Bowie EJ, Keene DR, 1220 1154 Stripp BR, Randell SH, Noble PW, Hogan BL: Type 2 alveolar cells 1221 1155 References are stem cells in adult lung. J Clin Invest 2013, 123:3025e3036 1222 1156 18. Chen F, Liu Z, Wu W, Rozo C, Bowdridge S, Millman A, Van 1223 1157 1. Wills-Karp M, Rani R, Dienger K, Lewkowich I, Fox JG, Perkins C, Rooijen N, Urban JF Jr, Wynn TA, Gause WC: An essential role for 1224 1158 Lewis L, Finkelman FD, Smith DE, Bryce PJ, Kurt-Jones EA, TH2-type responses in limiting acute tissue damage during experi- 1225 1159 Wang TC, Sivaprasad U, Hershey GK, Herbert DR: Trefoil factor 2 mental helminth infection. Nat Med 2012, 18:260e266 1226 1160 rapidly induces interleukin 33 to promote type 2 immunity during 19. Siracusa MC, Reece JJ, Urban JF Jr, Scott AL: Dynamics of lung 1227 1161 allergic asthma and hookworm infection. J Exp Med 2012, 209: macrophage activation in response to helminth infection. J Leukoc Biol 1228 1162 607e622 2008, 84:1422e1433 1229 1163 2. Van Dyken SJ, Nussbaum JC, Lee J, Molofsky AB, Liang HE, 20. Reece JJ, Siracusa MC, Southard TL, Brayton CF, Urban JF Jr, 1230 1164 Pollack JL, Gate RE, Haliburton GE, Ye CJ, Marson A, Erle DJ, Scott AL: Hookworm-induced persistent changes to the immunological 1231 1165 Locksley RM: A tissue checkpoint regulates type 2 immunity. Nat environment of the lung. Infect Immun 2008, 76:3511e3524 1232 1166 Immunol 2016, 17:1381e1387 21. Marsland BJ, Kurrer M, Reissmann R, Harris NL, Kopf M: Nippos- 1233 1167 3. Tjota MY, Hrusch CL, Blaine KM, Williams JW, Barrett NA, trongylus brasiliensis infection leads to the development of emphy- 1234 1168 Sperling AI: Signaling through FcRgamma-associated receptors on sema associated with the induction of alternatively activated 1235 1169 dendritic cells drives IL-33-dependent TH2-type responses. J Allergy macrophages. Eur J Immunol 2008, 38:479e488 1236 1170 Clin Immunol 2014, 134:706e713.e8 22. Martinez FO, Sica A, Mantovani A, Locati M: Macrophage activation 1237 1171 4. Tjota MY, Williams JW, Lu T, Clay BS, Byrd T, Hrusch CL, and polarization. Front Biosci 2008, 13:453e461 1238 1172 Decker DC, de Araujo CA, Bryce PJ, Sperling AI: IL-33-dependent 23. Lechner AJ, Driver IH, Lee J, Conroy CM, Nagle A, Locksley RM, 1239 1173 induction of allergic lung inflammation by FcgammaRIII signaling. J Rock JR: Recruited monocytes and type 2 immunity promote lung regen- 1240 1174 Clin Invest 2013, 123:2287e2297 eration following pneumonectomy. Cell Stem Cell 2017, 21:120e134.e7 1241 1175 5. Judd LM, Chalinor HV, Walduck A, Pavlic DI, Dabritz J, 24. Herold S, Mayer K, Lohmeyer J: Acute lung injury: how macrophages 1242 1176 Dubeykovskaya Z, Wang TC, Menheniott TR, Giraud AS: TFF2 orchestrate resolution of inflammation and tissue repair. Front Immu- 1243 1177 deficiency exacerbates weight loss and alters immune cell and cytokine nol 2011, 2:65 1244 1178 profiles in DSS colitis, and this cannot be rescued by wild-type bone 25. Mercurio AM, Rabinovitz I, Shaw LM: The alpha 6 beta 4 integrin and 1245 1179 marrow. Am J Physiol Gastrointest Liver Physiol 2015, 308:G12eG24 epithelial cell migration. Curr Opin Cell Biol 2001, 13:541e545 1246 1180 6. McBerry C, Egan CE, Rani R, Yang Y, Wu D, Boespflug N, Boon L, 26. Dhar DK, Wang TC, Maruyama R, Udagawa J, Kubota H, Fuji T, 1247 1181 Butcher B, Mirpuri J, Hogan SP, Denkers EY, Aliberti J, Herbert DR: Tachibana M, Ono T, Otani H, Nagasue N: Expression of cytoplasmic 1248 1182 Trefoil factor 2 negatively regulates type 1 immunity against Toxo- TFF2 is a marker of tumor metastasis and negative prognostic factor in 1249 1183 plasma gondii. J Immunol 2012, 189:3078e3084 gastric cancer. Lab Invest 2003, 83:1343e1352 1250

10 ajp.amjpathol.org - The American Journal of Pathology FLA 5.5.0 DTD Š AJPA2856_proof Š 13 March 2018 Š 3:29 pm Š EO: 2017_676