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Regulation of the Inflammatory Response: Enhancing Infiltration under Chronic Inflammatory Conditions

This information is current as Zhen Bian, YaLan Guo, Binh Ha, Ke Zen and Yuan Liu of October 1, 2021. J Immunol 2012; 188:844-853; Prepublished online 7 December 2011; doi: 10.4049/jimmunol.1101736 http://www.jimmunol.org/content/188/2/844 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Regulation of the Inflammatory Response: Enhancing Neutrophil Infiltration under Chronic Inflammatory Conditions

Zhen Bian,*,† YaLan Guo,* Binh Ha,* Ke Zen,† and Yuan Liu*

Neutrophil (polymorphonuclear leukocytes [PMN]) infiltration plays a central role in inflammation and is also a major of tissue damage. Thus, PMN infiltration must be tightly controlled. Using zymosan-induced as an in vivo PMN infiltration model, we show in this study that PMN response and infiltration were significantly enhanced in mice experiencing various types of systemic inflammation, including and diabetes. Adoptive transfer of leukocytes from mice with inflammation into healthy recipients or from healthy into inflammatory recipients followed by inducing peritonitis demonstrated that both circulating PMN and tissue were altered under inflammatory conditions and that they collectively contributed to enhanced PMN in-

filtration. Detailed analyses of dextran sulfate sodium-elicited colitis revealed that enhancement of PMN infiltration and macro- Downloaded from phage function occurred only at the postacute/chronic phase of inflammation and was associated with markedly increased IL-17A in serum. In vitro and ex vivo treatment of isolated PMN and macrophages confirmed that IL-17A directly modulates these cells and significantly enhances their inflammatory responses. Neutralization of IL-17A eliminated the enhancement of PMN infiltration and IL-6 production and also prevented severe tissue damage in dextran sulfate sodium-treated mice. Thus, IL-17A produced at the chronic stage of colitis serves as an essential feedback signal that enhances PMN infiltration and promotes inflammation. The

Journal of Immunology, 2012, 188: 844–853. http://www.jimmunol.org/

olymorphonuclear leukocyte (PMN) infiltration into tis- The mechanisms that control PMN response and regulate sues during inflammation plays a central role in innate their infiltration involve multiple cell types and various inflam- P . Upon or irritant infliction, matory factors, and studies in this area are still inadequate. local macrophages and other cells sense the insult and produce Some aspects of PMN function, such as and de- a panel of inflammatory mediators such as and che- granulation, have been studied in vitro by refined assays (1–3). mokines that stimulate the nearby microvasculature and attract However, these assays do not reveal the entire design of PMN large numbers of PMN to migrate across the vascular wall and inflammatory response and infiltration during inflammation and by guest on October 1, 2021 infiltrate into tissues. After arrival at the inflammatory site, PMN how these processes are dynamically regulated in vivo. In the perform and also release powerful antipathogen and study of PMN function in diabetes, we have constantly observed tissue-damaging reagents to kill and aberrant cells. that PMN isolated from diabetic patients showed impaired trans- Although these forceful PMN activities are extremely important migration in the in vitro chemotaxis assays (Y. Liu and K. Zen, for host defense, their adverse effects are also apparent, as dem- unpublished observations). These observations, which are con- onstrated in inflammatory bowel , , some car- sistent with other reports (4–6), indicated an important fact: diovascular conditions, inflammatory pulmonary and renal dis- that PMN from diabetic patients were different from those of eases, and viral/bacterial infection-associated damage. In these healthy donors; however, they could not ascertain whether the cases, the powerful but relatively low target specificity of PMN activities of PMN of diabetes were truly abated or, on the con- results in grave tissue/organ , often leading to a rapid trary, were primed (activated to an extent). In the second pos- progression of the pathophysiological condition. Thus, effective sibility, the primed PMN were hypersensitive and may have control of PMN infiltration would much alleviate the inflammatory become activated during the in vitro isolating procedure, re- condition. sulting in their inefficient chemotaxis in the later transmigra- tion assays (3). To further study PMN response and infiltration, we designed *Program of Cellular Biology and Immunology, Department of Biology, Georgia a two-layered in vivo inflammatory system in which a relatively State University, Atlanta, GA 30303; and †School of Life Sciences, Nanjing Univer- chronic inflammatory condition, such as colitis, type 1 diabetes, or sity, Nanjing, Jiangsu 210093, China other inflammatory condition, was first created in the experimental Received for publication June 16, 2011. Accepted for publication November 7, 2011. animals. After the condition was stabilized, a typical in- This work was supported in part by National Institutes of Health Grant DK62894 (to flammatory response, such as zymosan-induced peritonitis, was Y.L.) and a Research Scholar grant from the American Society (to Y.L.). induced, and the immediate PMN infiltration into the newly estab- Address correspondence and reprint requests to Dr. Yuan Liu, Program of Cellular lished inflammatory site was assayed within a few hours. Using Biology and Immunology, Department of Biology, Georgia State University, 161 Jesse Hill, 618 PSC, Atlanta, GA 30303. E-mail address: [email protected] this design, we found that active inflammation at the postacute/ chronic phase in particular induces systemic alterations in both Abbreviations used in this article: DiIC16(3), 1,1’-dihexadecyl-3,3,39,39-tetramethy- lindocarbocyanine iodide; DSS, dextran sulfate sodium; MPO, myeloperoxidase; PMN and cells within tissue microenvironments, including mac- PMN, polymorphonuclear leukocyte; rIL-17A, recombinant mouse IL-17A; STZ, rophages. Instead of suppressing inflammation, these changes re- streptozotocin. sult in an enhancement of PMN infiltration, hence exaggerating the Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 inflammatory reaction. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101736 The Journal of Immunology 845

Materials and Methods 3) was used to assess PMN chemotactic capability. Briefly, Mice and inflammatory mouse models leukocytes were isolated from femur bones of nontreated, healthy mice and mice with DSS-induced colitis or STZ-induced diabetes by flushing C57BL/6 mice 6–10 wk old (18–22 g) purchased from The Jackson the bone cavities using Hank’s buffer devoid of Ca2+ and Mg2+.After Laboratory (Bar Harbor, ME) were housed with free access to water and lysis of RBC, percentages of PMN in cell populations were determined food in a specific pathogen-free facility. All experiments using animals and by MPO assay and immunofluorescence staining using PE-Cy5–conju- procedures of animal care and handling were carried out following pro- gated anti-mouse Ly-6G (Gr-1) Ab. Aliquots of cell samples from each tocols approved by the Institutional Animal Care and Use Committee type of mice were also subject to further separation to obtain the pop- of Georgia State University. To establish dextran sulfate sodium (DSS)- ulation of mature PMN according to a protocol described by Dong and induced colitis (7, 8), 2% DSS (MP Biomedicals) prepared in pure water Wu (12). After normalization of the amount of mature PMN, the cells was given to the mice as the drinking water. Mice were inspected daily for (containing 2.5 3 106 PMN) were added into the upper chamber of the distress and colitis symptoms such as wet stool, diarrhea, bloody diarrhea, chemotaxis setup in 150 ml HBSS followed by inducing transmigration and weight loss. To induce hyperglycemia/type 1 diabetes (9, 10), the mice with 10 mM chemoattractant fMLF added into the lower chamber in 500 were fasted for 4 h and then injected with streptozotocin (STZ; Sigma- ml HBSS. The setup was then incubated at 37˚C, and the PMN trans- Aldrich) at a dose of 50 mg/kg body weight. STZ was freshly prepared in migrated across the filter into the lower chamber were then quantified at sodium citrate buffer (pH 4.5) prior to the injections, which were per- different time points by MPO assay. To test the effect of IL-17A on formed daily for 5 consecutive d. The mice were then maintained for an PMN, the freshly isolated cells loaded in the upper transmigration additional 2 wk to allow the diabetic condition to be stabilized. Serum chambers were treated with rIL-17A with or without neutralization Ab glucose levels were tested with the One-Touch glucose monitoring sys- or vehicle control buffer for 2 h before inducing transmigration by tem (LifeScan), and in general, .90% of injected mice developed hy- fMLF. perglycemia. To induce / inflammation, the mice were given LPS (Escherichia coli serotype 026:B6; Sigma-Aldrich), 0.3 mg/kg body Staining of tissue sections weight, in 70 ml PBS through the nares every other day for 15 d. For histochemical staining, mouse tissues including colon and lung were Downloaded from fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5–10-mm- Zymosan-induced peritonitis thin sections. Sections were stained with H&E. For staining of infiltrated This model was used to assay the acute PMN response and infiltration in vivo leukocytes and PMN, freshly sectioned tissues were blocked with 5% BSA (11). Briefly, mice were injected i.p. with 0.5 mg zymosan A (Sigma- followed by staining using a PE-Cy5–conjugated anti-mouse Ly-6G (Gr-1) Aldrich) that was prepared in 0.5 ml PBS. Mice were euthanized at vary- Ab. Images were then taken using an Olympus BX21 fluorescence mi- ing time points after the injection. PMN that transmigrated into the peri- croscope (Olympus).

toneum were collected by peritoneal lavage with 3 ml cold HBSS without http://www.jimmunol.org/ Ca2+ and Mg2+ followed by analysis with myeloperoxidase (MPO) assay, Cell labeling and Western blot cell counting, and immunofluorescence labeling using a PE-Cy5–conju- To directly observe IL-17A binding to PMN and macrophages, recombinant gated anti-mouse Ly-6G (Gr-1) Ab (eBioscience). Macrophages were an- murine IL-17A was labeled with FITC using an EZ-Label FITC alyzed using an Alexa Fluor 488-conjugated anti-mouse F4/80 Ab (clone labeling kit (Pierce). The FITC-labeled IL-17A was then incubated with BM8; BioLegend). For leukocyte adoptive transfer experiments, bone mar- freshly isolated mouse bone marrow leukocytes and peritoneal macrophages row cells were obtained by flushing femur bone cavities followed by la- for 30 min (25˚C) followed by washing and analysis by fluorescence mi- beling with the fluorescent cell tracer CFSE or 1,1’-dihexadecyl-3,3,39,39- croscopy. To detect IL-17A–induced protein phosphorylation changes in tetramethylindocarbocyanine iodide [DiIC16(3)] (both from Invitrogen). In macrophages, isolated murine peritoneal macrophages were treated with some experiments, the bone marrow cells were further separated by a Per- rIL-17A at concentrations of 0.5 and 2.0 ng/ml, or vehicle, for 48 h (37˚C). coll (GE Healthcare) density gradient to obtain the population of mature 6 6 + The cells were then lysed in a buffer containing 100 mM Tris (pH 7.5), 150 3 ∼ 3 by guest on October 1, 2021 PMN (12). In general, 5 10 labeled cells ( 2.5 10 were Gr-1 ) were mM NaCl, 1% Triton X-100, 1:100 dilution of a inhibitor mixture transfused into a recipient mouse via i.v. injection before inducing perito- (product code P8340; Sigma-Aldrich), and 3 mM PMSF followed by SDS- nitis using zymosan. To determine the role of IL-17A in PMN infiltration, PAGE and Western blot detection using the anti-phosphotyrosine Ab 4G10 ex vivo experiments were performed. In these experiments, freshly isolated (Millipore), an anti-phosphoserine Ab (Cell Signaling Technology), and an bone marrow cells were labeled with CFSE followed by treatment with anti- Ab (Sigma-Aldrich). a recombinant mouse IL-17A (rIL-17A) (2 ng/ml; BioLegend), with or without the presence of a neutralization anti–IL-17A Ab (low endotoxin, azide-free–purified anti-mouse IL-17A Ab [BioLegend]), or 50 ml serum collected from nontreated mice or mice treated with DSS for 12 d. After Results incubation for 2 h (37˚C), the cells (∼2.5 3 106 PMN) were combined with Analysis of the acute PMN response in vivo by equal amount of DiIC16(3)-labeled bone marrow PMN before transfer into zymosan-induced peritonitis healthy recipient mice. Zymosan-induced peritonitis was then performed to test the transferred PMN infiltration into the . In this study, we used zymosan-induced peritonitis (11), a com- monly used self-resolving acute inflammation model, to assay the Measurement of cytokines PMN immediate response and infiltration in vivo. For these ex- Whole blood from mice was collected without anticoagulation additives, periments, healthy C57BL/6 mice that were maintained in a and the serum was isolated. For ELISA, mouse serum of different dilutions pathogen-free animal facility were injected i.p. with zymosan A, was incubated in anti- Ab-coated wells in 96-well plates for 2 h followed by analysis for PMN recruitment into the peritoneum (25˚C). After washing, the wells were incubated with biotin-conjugated after various time periods. As shown in Fig. 1A and Table I, before detecting Abs and HRP-conjugated streptavidin followed by detection with zymosan injection, ∼2.6 3 106 cells were present in the perito- o-phenylenediamine dihydrochloride (Sigma-Aldrich) substrate. Both ∼ + capture and detecting Abs against different cytokines including IL-1b, neum, and among these cells, 30% were F4/80 macrophages, + IL-4, IL-6, IL-12 p40/70, IL-17A, IFN-g, TNF-a, MCP-1, IL-10, and GM- and the remainder contained few (∼5%) Gr-1 , which presumably CSF were purchased from BD Biosciences and BioLegend. Purified re- were tissue and other tissue cells. Soon after zy- combinant murine cytokines used as the standards were purchased from mosan challenge, PMN began to infiltrate into the peritoneum, PeproTech. To detect cytokines produced by in vitro-treated macrophages, resulting in drastic changes in the cell composition and numbers. peritoneal macrophages were obtained by lavage of the peritoneal space 6 and collection by centrifugation. The macrophages were then stimulated As shown, at 2 h, small numbers of PMN (∼1 3 10 ) trans- with zymosan or rIL-17A (2 ng/ml) with or without the presence of the migrated across the and arrived at the peritoneum. anti–IL-17A Ab (2 mg/ml) for various time periods up to 48 h in DMEM Meanwhile, the majority of macrophages were seemingly leaving with 8% FBS (37˚C). After the treatment, the supernatants were collected, the area, resulting in a significant decrease of the F4/80+ cells and levels of IL-6 and other cytokines were detected by ELISA. (Table I). Massive PMN infiltration into the peritoneum occurred In vitro PMN chemotaxis assay after 2 h, resulting in ∼107 PMN and .1.2 3 107 PMN recruited An in vitro transmigration setup using transwell devices with - to the location at 4 and 6 h, respectively. By 8 h, PMN infiltration coated transfilters (0.33 cm2,5mm pore size) established previously (1– slowed rapidly or stopped, resulting in a decrease in the number of 846 ENHANCED NEUTROPHIL INFILTRATION DURING

Enhancing PMN infiltration under chronic inflammatory conditions To investigate whether chronic inflammation affects PMN infil- tration, we induced various inflammatory conditions in C57BL/6 mice before performing the zymosan-peritonitis assay. In these experiments, mice were treated with 2% DSS to induce colitis (7, 8), STZ to induce type 1 diabetes (9, 10), or LPS to induce bronchitis/lung inflammation (15). A group of mice were also treated with one dose of emulsified CFA (50% in PBS) to induce chronic inflammation. As shown in Fig. 2A, progressive body weight loss, which was associated with diarrhea and colitis, was observed in DSS-treated mice. Staining of colon tissues after treating the mice with DSS demonstrated significant intestinal inflammation that was associated with aggressive leukocyte in- filtration, severe mucosal damage, and loss of epithelial structures (Fig. 2A). Treating mice with STZ and LPS also successfully in- duced diabetic and lung inflammatory conditions. As shown in Fig. 2B, all of the STZ-treated mice developed hyperglycemia,

with the mean blood glucose level being increased to 431.7 6 56.1 Downloaded from mg/dl, whereas the control group treated with vehicle (PBS) 6 FIGURE 1. Inflammatory response/peritonitis after zymosan challenge. maintained a blood glucose level of 141.2 14.0 mg/dl. Mean- Mice receiving injection (i.p.) of zymosan A were euthanized at the in- while, treating mice with low-dose LPS via the nares for 15 d dicated time points. A, Macrophages and PMN lavaged from the peri- induced evident upper respiratory inflammation that was charac- toneum were stained with anti-F4/80 (green) and anti–Gr-1 (red) Abs, terized by noticeable leukocyte infiltration around bronchi, bron-

respectively, and visualized under a fluorescent microscope. B, Serum con- chioles, and in some area of the lung (Fig. 2C). http://www.jimmunol.org/ centrations of inflammatory cytokines during peritonitis were evaluated by We then examined PMN function in these mice with various 6 , ELISA. Data are expressed as means SEM; n = 6 mice/group. **p inflammatory conditions by performing zymosan-induced perito- 0.01 versus respective serum cytokine levels at 0 h. nitis. For these experiments, we used the mice that had been treated with DSS for 12 d and manifested apparent colitic condition. As shown in Fig. 2D, compared with the control healthy mice that PMN in the area. After the peak of PMN recruitment, PMN just began to infiltrate into the peritoneum at 2 h and only infiltration began to increase at 6 h and continued to increase small numbers (∼1 3 106 PMN) were detected, significantly in- thereafter, suggesting that the acute inflammation proceeded to creased numbers of PMN (.6 3 106) were recruited to the area in a resolving stage. The infiltrating and their mediated colitic mice at the same time point. Similar early promoted PMN by guest on October 1, 2021 clearance likely accounted for the decrease of PMN numbers in infiltration into the peritoneum in response to zymosan challenge the peritoneum at later time points. Consistent with other reports was also observed in STZ-induced diabetic mice and LPS-treated (13, 14), we detected a robust but transient increase in IL-6 in the mice. These mice were used to test for PMN infiltration by peri- peritoneal fluid and in the blood serum (Fig. 1B, Table II) after tonitis after the STZ- and LPS-induced pathological conditions zymosan challenge. As observed, the time course of IL-6 change were stabilized. As shown in Fig. 2D, at 2 h, 5.6 3 106 PMN and tightly correlated with the dynamics of PMN infiltration, sug- ∼3 3 106 PMN were recruited to peritonea in STZ- and LPS- gesting a potential role of this cytokine in controlling the episode treated mice, respectively. We also induced peritonitis in the mice of zymosan-induced peritonitis. Moderate increases in IL-1, IL- that were treated with CFA 1 mo ago. As shown in Fig. 2D, this 17A, and TNF-a were also detected. The dynamics of IL-1 re- form of chronic inflammation also significantly enhanced PMN sembled that of IL-6, but IL-17A was only increased at later time response to zymosan, resulting in .7 3 106 PMN being recruited periods, when the massive PMN transmigration was nearly com- to the peritoneum by 2 h. pleted (Fig. 1B, Table II). Kinetic analysis of PMN infiltration (Fig. 2E) in colitic mice by plotting the total numbers of PMN transmigrated as a function of time showed that the robust PMN infiltration occurred earlier than that in the control mice. Indeed, .70% of the infiltrated PMN in Table I. Leukocytes in the peritoneum and their dynamic changes during zymosan-induced peritonitis mice with DSS treatment for 12 d had arrived at the peritoneum by 2 h, whereas in the controls, the majority of PMN were Cell Number (Mean 6 SEM) recruited by 4 h. Significant enhanced PMN infiltration into the peritoneum at an early time point (2 h) was also observed in mice + 3 5 + 3 5 3 5 Time (h) F4/80 ( 10 ) Gr-1 ( 10 ) Total ( 10 ) with DSS treatment for 9 d (Fig. 2E). The kinetic results also 0 10.4 6 1.4 2.0 6 0.7 26.4 6 3.7 indicated that the early enhancement of PMN infiltration in col- 2 1.5 6 0.2 10.7 6 1.5 15.3 6 2.2 itic mice was not due to inflammation-induced increases in PMN 4 0.9 6 0.1 96.2 6 6.8 120.5 6 8.5 6 1.5 6 0.2 124.3 6 10.4 146.2 6 12.2 production but to accelerated PMN response and transmigration. 8 8.2 6 1.1 115.1 6 6.6 154.3 6 8.8 In fact, the accumulative PMN numbers that infiltrated into the peritoneum in colitic mice at 4 h were not higher, but tended to be Mice injected with zymosan A were euthanized at various time points followed by collection cells from peritonea and staining for macrophages and PMN/granulocytes lower, than those in the controls. Parallel analyses of PMN using an anti-F4/80 Ab (green) and an anti–Gr-1(red) Ab, respectively. Control cell numbers in the peripheral circulation and in bone marrow indi- staining used normal rat serum. The cells of different staining were counted and analyzed by FACS. The 0 h time point was determined without zymosan injection. cated that despite the increase of GM-CSF in the serum (Table The table shows the mean cell number 6 SEM, n = 6 mice/group. III), continual DSS treatment ($9 d) and colitis exhausted the The Journal of Immunology 847

Table II. Time-course detection of serum cytokines during zymosan A-induced peritonitis

Time Post-Zymosan Injection (h)

Cytokine (pg/ml) 0 2 4 6 8 TNF-a 49.2 6 10.3 118.1 6 20.6* 111.8 6 16.0* 96.4 6 12.8* 100.7 6 16.5* IL-1b 31.0 6 6.6 159.3 6 26.5* 498.0 6 58.9** 113.7 6 32.5* 64.3 6 22.7 IL-6 65.6 6 22.8 2180.0 6 387.9** 4713.1 6 1333.4** 1023.7 6 322.4** 555.0 6 165.6** IL-17A 109.6 6 10.8 126.9 6 12.7 370.3 6 94.5** 562.0 6 77.4** 517.3 6 64.6** Concentrations of inflammatory cytokines in serum during peritonitis were evaluated by ELISA. Data are expressed as means 6 SEM; n = 6 mice/ group. *p , 0.05, **p , 0.01 versus respective serum cytokine levels at 0 h.

entire system, and also consumed PMN availability in the cir- designed two directional, adoptive leukocyte transfer and perito- culation (data not shown). nitis experiments. In these, donor PMN derived from mice with different inflammatory conditions were transfused into healthy, Inflammation-induced modulations of both circulating PMN noninflammatory recipient mice and tested for response and in- and tissue microenvironments contribute to the promotion of filtration in zymosan-induced peritonitis; conversely, donor PMN PMN infiltration

from healthy mice were transfused into recipient mice with ongoing Downloaded from The results of enhanced PMN infiltration in mice under various inflammation and then tested for infiltration in response to zymosan inflammatory conditions suggest a likelihood that alterations oc- challenging. The former experiments determine whether chronic curred in either PMN, which directly promote PMN transmigra- inflammation converts PMN to be faster migrating cells (even in a tion, or in cells within the peritoneal vicinity, which led to an ear- new host environment). The latter experiments examine the peri- lier local response to zymosan and/or contributed to the facilitation toneal tissue microenvironment and determine whether chronic when PMN transmigrated through. To test these possibilities, we inflammation could induce changes that promote PMN infiltration http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 2. Mice with various chronic inflammatory conditions display enhanced PMN infiltration during zymosan-induced peritonitis. A, DSS-induced colitis. C57BL/6J mice were given 2% DSS in drinking water continuously, and development of colitis was monitored by body weight loss, diarrhea, etc., and confirmed by of the colonic tissues. B, STZ-induced type 1 diabetes. Mice were consecutively injected with STZ or the vehicle (CTL) for 5 d. Serum glucose levels were assayed 3 wk after the injections. C, Inducing upper respiratory inflammation by LPS. LPS or vehicle was given to mice via the nares every other day for 15 d. The figure shows the histology images (H&E staining) of lung tissue sections after vehicle (Ctl.) or LPS treatment. D and E, Enhanced PMN infiltration in mice with systemic inflammation. Control healthy mice and mice with colitis (DSS for 12 d), hyperglycemia (3 wk post-STZ treatment), or lung inflammation (LPS) were tested for PMN infiltration by zymosan A-induced peritonitis. Mice that received one dose of emulsified CFA (50% in PBS) were also tested in the assay. D and E show the numbers of PMN infiltrated into the peritoneum at 2 h and the kinetics of PMN infiltration into the peritoneum, respectively. The data represent one of eight independent experiments and are expressed as means 6 SEM; n = mice number/group. **p , 0.01, ***p , 0.001 versus respective controls. 848 ENHANCED NEUTROPHIL INFILTRATION DURING INFLAMMATION

Table III. Serum cytokine levels during DSS-induced colitis

DSS Treatment

Cytokine (pg/ml) Day 0 Day 3 Day 6 Day 9 Day 12 TNF-a 52.8 6 5.6 71.0 6 5.8* 69.7 6 8.1 57.8 6 11.6 52.1 6 9.3 IL-1b 35.4 6 8.6 28.5 6 6.0 39.4 6 11.2 227.5 6 22.7** 144.6 6 16.4** IL-6 70.2 6 17.6 78.7 6 10.4 132.2 6 42.4* 430.8 6 109.1** 173.4 6 24.0** IL-17A 113.6 6 10.6 108.9 6 10.9 249.8 6 24.1* 714.1 6 93.6** 2023.9 6 172.1*** MCP-1 30.3 6 8.6 37.2 6 9.4 156.8 6 16.2** 79.4 6 18.2* 22.5 6 4.3 IFN-g 51.0 6 6.7 44.2 6 7.8 51.1 6 9.3 40.4 6 18.0 41.2 6 6.4 IL-12/23(p40) 55.6 6 7.3 57.8 6 6.5 77.4 6 11.9* 67.0 6 6.1 54.6 6 7.2 IL-4 39.5 6 4.2 43.6 6 7.3 ND ND ND IL-10 59.2 6 20.9 133.6 6 16.4* 35.5 6 12.8 30.4 6 11.3 27.2 6 13.6 GM-CSF ND 65.0 6 16.3* 29.4 6 9.2 21.0 6 8.4 ND Inflammatory cytokine levels in serum during DSS-induced colitis were evaluated by ELISA. Data are expressed as means 6 SEM; n = 6-8 mice/ group. ND, undetectable. *p , 0.05, **p , 0.01, ***p , 0.001 versus respective controls. during peritonitis. Control experiments were performed by trans- Fig. 3A shows the results of the transfusion of PMN from in- fusion of PMN from healthy mice into other healthy mice followed flammatory mice into healthy recipients and the infiltration of by testing the donor PMN infiltration. donor PMN under zymosan challenge. As shown, although the Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. Systemic inflammation alters both PMN and tissue microenvironments. A, Testing donor PMN from inflammatory mice response in healthy recipients by zymosan-peritonitis. Bone marrow cells (2.5 3 106 mature PMN) derived from colitic (DSS, 12 d), diabetic (STZ), or control healthy mice were labeled with CFSE and transfused into healthy recipients, respectively. Figure shows the microscopic analysis of infiltrated donor PMN in peritoneal lavage after inducing peritonitis for 2 h and the total numbers of donor PMN infiltrated at 2 and 4 h. The result represents one of four independent experiments. B,PMN chemotaxis in vitro. Bone marrow leukocytes from healthy control and colitic (DSS, 12 d) mice were normalized for equal PMN contents by MPO assays before being applied into the upper chambers of the transmigration setups. Chemotaxis induced by fMLF was measured at 45 min. C, Cotransfer of leukocytes from healthy and inflammatory mice and testing by peritonitis. A mixture of bone marrow leukocytes derived from healthy mice (red, DiIC16 labeling) and colitic mice (DSS, 12 d) (green, CFSE labeling) (2.5 3 106 PMN each) were transferred into the same healthy recipients. The images show zymosan-induced PMN infiltration into the peritoneum at 2 h in the peritoneal lavage. The result represents one of five independent experiments. D, Transfer of PMN from healthy mice into inflammatory recipients followed by testing PMN infiltration during peritonitis. Bone marrow leukocytes from five healthy mice were combined and labeled, followed by transfusion into different types of inflammatory recipient mice (three mice per type, 2.5 3 106 PMN/mouse). E, Peritoneal macrophages obtained by lavaging peritonea were stimulated in vitro using zymosan (1 mg/ml) in DMEM (containing 5% FBS) at 37˚C. IL-6 secreted into the medium at different time points was assayed by ELISA. The data are expressed as means 6 SEM. *p , 0.05, **p , 0.01 versus respective controls. The Journal of Immunology 849 host mice had no prior inflammation, donor PMN from colitic and rophages in the peritoneum of STZ-induced diabetic mice (data diabetic mice (Fig. 3A, labeled with CFSE, green) still displayed not shown). Thus, these results confirmed tissue environment much faster infiltration, resulting in more cells transmigrated into changes under chronic inflammation. Increases in cytokine pro- the peritoneum at 2 h than control donor PMN from healthy mice. duction from the local macrophages certainly contributed to en- In parallel, we also performed in vitro chemotaxis assays and hanced PMN infiltration. compared the transmigration of PMN derived from DSS-treated mice and from healthy mice. To avoid activation of PMN, espe- Role of IL-17 in colitis-associated inflammatory modulation cially those from mice with inflammation, we directly applied the The findings that circulating PMN and peritoneal macrophages had total bone marrow leukocytes without further isolation. As shown altered responsiveness under colitis and diabetes were striking in Fig. 3B, greater numbers of PMN transmigrating across matrix- given that both cells were distal to the original inflammatory sites in coated filters toward the chemoattractant fMLF were observed in those conditions. We thus hypothesized that critical inflamma- samples derived from DSS-treated mice than those from healthy tory factors were released into the circulation, through which it mice. In Fig. 3C, we further labeled donor PMN or total bone reached and regulated cells at a distance. To identify these impor- marrow cells derived from DSS-treated mice and from healthy tant factors, especially those that might contribute to enhance PMN mice with different dyes (green and red) and simultaneously trans- infiltration, we analyzed inflammatory cytokines associated with fused into the same recipient mice (at a 1:1 ratio of the PMN DSS-induced colitis in the serum. As shown in Table III, whereas number). As shown, zymosan induced more infiltration of PMN most of cytokines tested were reported in colonic tissues (7, 16– from colitic mice (green) than those from noncolitic, healthy mice 19), these cytokines stayed at low or moderate levels in the serum (red) at 2 h, confirming that PMN in chronic inflammatory mice throughout the course of the DSS treatment. The only cytokine had an enhanced ability to respond and migrate toward inflam- that we found significantly elevated in the serum was IL-17A (Fig. Downloaded from matory stimuli. 4A), which started to increase at days 6–8 of DSS treatment and To investigate whether the tissue microenvironment also con- continued increasing, eventually reaching .2 ng/ml. We found tributed to the enhanced PMN infiltration under inflammatory that not only was its elevation in the serum extraordinary, but also conditions, we transferred the PMN of healthy mice into DSS- and that the dynamics of IL-17A tightly correlated with the en- STZ-treated chronic inflammatory mice (recipients). Inducing peri- hancement of PMN infiltration observed in peritonitis. As shown tonitis subsequently showed significantly faster infiltration of do- in Fig. 4B, inducing peritonitis in mice that were treated with http://www.jimmunol.org/ nor PMN in mice with ongoing colitis or diabetes than in control DSS for different days revealed that significant promotion of healthy recipients (Fig. 3D). These results suggest that consider- PMN infiltration occurred only after DSS treatment for 6 d and able alterations had occurred in the peritoneal tissue environment continued to be enhanced thereafter. Supporting this observation, in inflammatory mice and that these changes promoted PMN in- assay of peritonitis-associated IL-6 release also demonstrated that filtration. Because peritoneal macrophages play an important role enhancement of IL-6 release upon zymosan challenging occurred in the development of peritonitis, we collected these macrophages in mice with DSS treatment for .6 d (Fig. 4C). Interestingly, and directly tested their responses to zymosan in vitro. As shown significant body weight decrease in DSS-treated mice also started in Fig. 3E, in the cell-culture dishes, zymosan induced increased at approximately the same time (Figs. 2A,5C). These results by guest on October 1, 2021 IL-6 production in macrophages collected from DSS- and STZ- suggest that, although DSS-induced colitis started much earlier treated mice than those isolated from noninflammatory controls. than day 6, as elevations of inflammatory cytokines (Table III) Interestingly, we observed significantly increased numbers of mac- and leukocyte infiltration into the colonic tissues were apparent on

FIGURE 4. Enhancement of PMN infiltration in DSS-induced colitis is correlated with IL-17A production. A, Time-course analysis of serum cytokines during DSS-induced colitis (also see Table III). B, Analysis of PMN infiltration by zymosan-induced peritonitis in mice treated with DSS for different days. Note that significantly enhanced PMN infiltration occurs after 6 d of DSS treatment and in parallel with increases in IL-17A. C, Analysis of peritonitis- associated IL-6 production in mice treated with DSS for different days. D, Tissue section of colon intestines and labeling for infiltrated PMN using an anti– Gr-1 Ab. The data represent one of at least three independent experiments and are expressed as means 6 SEM with at least five mice per group. *p , 0.05, **p , 0.01, ***p , 0.001 versus respective controls. 850 ENHANCED NEUTROPHIL INFILTRATION DURING INFLAMMATION days 2–5 (Fig. 4D), the quick collapse of the pathological con- under inflammatory conditions, we tested whether IL-17A also dition at the later stage was associated with the enhancement of directly modulates function. As shown in Fig. 6C, PMN infiltration and the consequent severe tissue damage. treatment of peritoneal macrophages isolated from healthy mice To further explore the role of IL-17A in the enhancement of with rIL-17A markedly elevated IL-6 production upon the sub- PMN infiltration, we treated the DSS mice with a functional neu- sequent stimulation with zymosan. Western blot analyses of IL- tralization Ab against IL-17A (20, 21) (i.v.). As shown in Fig. 5, 17A–treated macrophages indicated significantly increased pro- suppression of IL-17A function by the Ab in DSS-treated mice tein tyrosine phosphorylations (Fig. 6D), suggesting that active nearly completely eliminated the enhanced PMN infiltration at 2 h signaling events were elicited by IL-17A. In addition, we also during zymosan-induced peritonitis (Fig. 5A) and such event- observed direct binding of rIL-17A to the cell surfaces of PMN associated increases in IL-6 (Fig. 5B). The colitic symptoms in and macrophages (not shown), presumably to the receptors. In these DSS-treated mice, such as body weight loss, diarrhea, and conclusion, these results strongly suggest that IL-17A, which was intestinal tissue damage, were also largely alleviated (Fig. 5C). produced at a large amount in the postacute/chronic stage of co- However, IL-17A neutralization had no effect on the basal level of litis, serves as a critical feedback factor and contributes to rein- PMN infiltration, as large numbers of PMN were still recruited forcement of the inflammatory reaction through systemically into the peritoneum at 4 h during peritonitis even with the Ab modulating leukocytes in the circulation and tissues. administration (Fig. 5A). PMN infiltration into intestinal mucosa was also present in DSS-treated mice with anti–IL-17A adminis- Discussion tration, but remained at a low level (data not shown). We further PMN, which comprise more than two-thirds of peripheral leuko- performed ex vivo experiments and tested whether exposure to cytes, are the first-tier responsive cells during inflammation. As IL-17A directly modulates PMN function. In these experiments, shown in zymosan-induced peritonitis, when a stimulatory signal is Downloaded from bone marrow leukocytes isolated from healthy mice were ex vivo effectively produced, PMN rapidly transmigrate through the vas- treated with a functional rIL-17A and then transferred into other culature and tissues and arrive at the inflammatory site within healthy mice (recipients) followed by testing by zymosan-induced a few hours. After a short period and vigorous activity, PMN in- peritonitis. As shown in Fig. 6A, PMN treated with rIL-17A filtration is promptly ceased, provided that the inflammatory causes demonstrated markedly accelerated infiltration into the perito- are largely cleared, to confine and limit tissue damage. However, neum during peritonitis compared with nontreated control PMN when the inflammatory causes linger, as those in DSS-induced http://www.jimmunol.org/ that were cotransferred into the same recipients. As expected, the colitis, PMN infiltration continues, and the inflammatory reac- presence of the anti–IL-17A Ab in the treatment completely tion prolongs and becomes chronic. In this study, we employed eliminated the enhancement of PMN infiltration. Similarly, PMN inflammation mouse models and studied PMN infiltration under that were treated with the serum from colitic mice (DSS treatment various chronic conditions. We found that animals that were for 12 d) also manifested accelerated infiltration in recipient mice preconditioned with nonresolving colitis, type 1 diabetes, or other during peritonitis. Consistent with these results, assaying PMN inflammatory conditions had significantly enhanced PMN infil- chemotaxis in vitro (Fig. 6B) observed significantly augmented tration during zymosan-induced peritonitis. Cross-transfer of leu- chemotactic transmigration for PMN treated with rIL-17A, kocytes from mice with ongoing inflammation to healthy recipients, by guest on October 1, 2021 whereas neutralization of IL-17A function erased such augmen- or from healthy mice to inflammatory recipients, further revealed tation. Because tissue macrophage responses were also altered that both circulating PMN and tissue microenvironments were

FIGURE 5. Suppression of IL-17A function by a neutralization Ab eliminates the augmentation of inflammation and PMN infiltration at postacute stage of DSS-induced colitis. Mice under DSS treatment were administered (i.v.) 30 mglow endotoxin, azide-free–purified anti-mouse IL-17A Ab on days 4, 6, 8, and 10. Control injections were performed by administration of an - matched Ab (rat IgG1) (BioLegend). On day 12, mice with and without Ab administration were tested by zymosan-induced peritonitis. A, PMN infiltration into the peritoneum at 2 and 4 h. B, Serum IL-6 after zymosan challenge. C, The body weight of mice treated with free water or DSS plus Abs. The data are expressed as means 6 SEM and represent one of at least three inde- pendent experiments with at least five mice per group. **p , 0.01, ***p , 0.001 versus re- spective controls. The Journal of Immunology 851

FIGURE 6. IL-17A directly enhances PMN and macrophage inflammatory functions. A, PMN ex vivo exposure to IL-17A exhibits an accelerated in- filtration during zymosan-induced peritonitis. Bone marrow leukocytes from healthy mice were treated for 2 h with an rIL-17A (2 ng/ml) or serum from mice with DSS-induced colitis (DSS, 12 d) in the presence or absence of the neutralization anti–IL- 17A Ab. After treatment, the cells were labeled with CSFE (green) and cotransferred with control non- treated bone marrow leukocytes (red) into healthy recipient mice followed by testing for PMN infil- tration during zymosan-induced peritonitis. B, IL- 17A directly enhances PMN chemotactic transmi- gration. Bone marrow PMN with and without exposure to IL-17A were assayed in vitro for chemo- taxis toward fMLF for 90 min. C, IL-17A promotes

IL-6 production in macrophage upon stimulation. Downloaded from Peritoneal macrophages obtained from healthy mice were treated with rIL-17A (2 ng/ml) with or without the presence of anti–IL-17A Ab for 48 h (37˚C). IL-6 production in cells with or without zymosan stimu- lation (2 h) was assayed by ELISA. D, Western blot analyses of protein phosphorylation of macrophages treated with IL-17A. **p , 0.01. http://www.jimmunol.org/

altered under chronic inflammatory conditions and that these proinflammatory cytokine IL-6. Peritoneal macrophages isolated changes collectively contributed to the promotion of PMN infil- from late-stage colitic mice also demonstrated enhanced tration during peritonitis. responses upon inflammatory challenge. All of these results to- by guest on October 1, 2021 Detailed analysis of DSS-induced colitis found that PMN in- gether indicate that when inflammation prolongs, feedback sig- filtration into intestinal mucosa occurred shortly after the starting nals are produced and trigger a systemic regulation of the entire of DSS treatment (days 2 to 3); concomitantly, a panel of in- inflammatory reaction. In the case of DSS-induced colitis, large flammatory cytokines associated with colitis, including low levels amounts of IL-17A produced at the postacute stage prime cir- of IL-17A, which are presumably released from the local gd culating PMN and tissue macrophages and enhance their in- T cells and infiltrated PMN (22), were also produced (Table III). flammatory functions. These regulations by IL-17A, which are However, when the DSS treatment continued over 6–8 d, we presumably designed to accelerate the removal of the original observed extraordinarily high levels of IL-17A in the blood se- inflammatory cause, nevertheless lead to exaggerated inflam- rum. Our studies suggest that this late-produced strong wave of mation-associated tissue damage. Suppression of IL-17A–me- IL-17A, which was likely through the activation of the second- diated propagation of inflammation mitigates the con- tier inflammatory cells such as Th17 cells, is capable of reaching dition. and regulating cells distant from the original inflammatory site Priming PMN under inflammatory conditions and by inflam- through circulation, leading to systemic modulations of the en- matory factors has been observed in research through the years tire inflammation and homeostasis. Indeed, our data showed that and has been considered to be a major factor responsible for the both macrophages and PMN in distant tissues are regulated by of organ failure occurred under postinjury conditions such released, circulating IL-17A. In fact, PMN appeared to such as trauma, , and and other conditions such as adult be altered even before their release from the bone marrow, respiratory distress syndrome, , cystic fibrosis, blood trans- consistent with the microarray study by Theilgaard-Monch et al. fusion, etc. (25–32). Reported agents that prime PMN include (23, 24) in which various cytokine receptors including IL-17R -activating factor (33, 34), G-CSF (35, 36), GM-CSF (37), were identified in bone marrow myelocytes, especially mature IL-1b (38), IL-6 (39), IL-8 (35), etc. In this study, our data suggest PMN. Strikingly, the time course of elevation of IL-17A in se- that IL-17A is likely to be a critical priming agent for PMN and rum during DSS treatment tightly correlated with the significant macrophages under inflammatory conditions, yet the mechanism enhancement of PMN infiltration observed in zymosan-induced underlying its effect and the phenotypic characteristics of PMN peritonitis. Subsequent in vitro and ex vivo experiments con- (40) after exposure to IL-17A need to be further determined. firmed that IL-17A directly modulates PMN and significantly Although many studies have shown that IL-17A production is accelerated PMN chemotactic transmigration toward inflamma- regulated by IL-6 and IL-23 (and other cytokines) (41, 42), our tory stimuli. Not only modulating PMN, IL-17A also enhances study in DSS-induced colitis only detected slight increases of the responses and sensitivities of macrophages around the body. these cytokines in the serum, implicating that IL-6– and IL-23– In vitro exposure of macrophages to IL-17A prior to stimula- mediated activation of Th17 cells may be mainly restricted in the tion with zymosan resulted in much increased production of the local intestinal tissues. In addition, previous studies by Schwar- 852 ENHANCED NEUTROPHIL INFILTRATION DURING INFLAMMATION zenberger et al. (43, 44) and others (45, 46) have shown that 15. Szarka, R. J., N. Wang, L. Gordon, P. N. Nation, and R. H. Smith. 1997. A increases of IL-17A promote promyelocyte proliferation and PMN murine model of pulmonary damage induced by via intra- nasal instillation. J. Immunol. Methods 202: 49–57. maturation. Because IL-17A was significantly increased in DSS- 16. Yan, Y., V. Kolachala, G. Dalmasso, H. Nguyen, H. Laroui, S. V. Sitaraman, treated mice, it is thus likely that DSS-induced colitis was asso- and D. Merlin. 2009. Temporal and spatial analysis of clinical and molec- ular parameters in dextran sodium sulfate induced colitis. PLoS ONE 4: ciated with increased and PMN production, which e6073. are important for the ongoing inflammation and continual PMN 17. Alex, P., M. Ye, N. C. Zachos, J. Sipes, T. Nguyen, M. Suhodrev, L. Gonzales, infiltration into intestines. However, increases of PMN production Z. Arora, T. Zhang, M. Centola, S. E. Guggino, and X. Li. 2010. 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