We investigated the role of interleukin-17 (IL-17)/IL-17 receptor (IL-17R)-mediated signaling in the protective immunity against Toxoplasma gondii. IL-17R−/− mice developed a normal adaptive immunity against the parasite. However, increased mortality in the knockout animals can be attributed to a defect in the migration of polymorphonuclear leukocytes to infected sites during early infection

Happel, Kyle

Kelly, Michelle N

Kolls, Jay K

Schwartzman, Joseph D

English

Dartmouth Digital Commons (Dartmouth College)

Dartmouth College Dartmouth Digital Commons Open Dartmouth: Published works by Dartmouth faculty Faculty Work 1-2005 Interleukin-17/Interleukin-17 Receptor-Mediated Signaling Is Important for Generation of an Optimal Polymorphonuclear Response against Toxoplasma gondii Infection Michelle N. Kelly Louisiana State University Health Sciences Center New Orleans Jay K. Kolls University of Pittsburgh Kyle Happel Louisiana State University Health Sciences Center New Orleans Joseph D. Schwartzman Dartmouth College Follow this and additional works at: https://digitalcommons.dartmouth.edu/facoa  Part of the Infectious Disease Commons, Medical Immunology Commons, Medical Microbiology Commons, and the Parasitic Diseases Commons Dartmouth Digital Commons Citation Kelly, Michelle N.; Kolls, Jay K.; Happel, Kyle; and Schwartzman, Joseph D., "Interleukin-17/Interleukin-17 Receptor-Mediated Signaling Is Important for Generation of an Optimal Polymorphonuclear Response against Toxoplasma gondii Infection" (2005). Open Dartmouth: Published works by Dartmouth faculty. 953. https://digitalcommons.dartmouth.edu/facoa/953 This Article is brought to you for free and open access by the Faculty Work at Dartmouth Digital Commons. It has been accepted for inclusion in Open Dartmouth: Published works by Dartmouth faculty by an authorized administrator of Dartmouth Digital Commons. For more information, please contact dartmouthdigitalcommons@groups.dartmouth.edu. INFECTION AND IMMUNITY, Jan. 2005, p. 617–621 Vol. 73, No. 10019-9567/05/$08.000 doi:10.1128/IAI.73.1.617–621.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.Interleukin-17/Interleukin-17 Receptor-Mediated SignalingIs Important for Generation of an OptimalPolymorphonuclear Response againstToxoplasma gondii InfectionMichelle N. Kelly,1 Jay K. Kolls,2 Kyle Happel,3 Joseph D. Schwartzman,4Paul Schwarzenberger,5 Crescent Combe,1 Magali Moretto,1and Imtiaz A. Khan1*Department of Microbiology, Immunology and Parasitology,1 Department of Pulmonary Medicine,3and Division of Gene Therapy,5 Louisiana State University Health Sciences Center,New Orleans, Louisiana; Department of Pediatrics, University of Pittsburgh,Pittsburgh, Pennsylvania2;and Department of Pathology, DartmouthMedical School, Lebanon, New Hampshire4Received 15 April 2004/Returned for modification 7 June 2004/Accepted 8 September 2004We investigated the role of interleukin-17 (IL-17)/IL-17 receptor (IL-17R)-mediated signaling in the pro-tective immunity against Toxoplasma gondii. IL-17R/ mice developed a normal adaptive immunity againstthe parasite. However, increased mortality in the knockout animals can be attributed to a defect in themigration of polymorphonuclear leukocytes to infected sites during early infection.While the induction of a cell-mediated response is essentialfor protection against Toxoplasma gondii, the initial innateimmune response led by neutrophils has also been reported tobe critical for successful resolution of the infection (1, 6, 13).Depletion studies have shown that their loss leads to exacer-bation of infection (3, 13). The factors involved in the devel-opment of this neutrophil response against infection have notbeen well studied. Recently, interleukin 17 (IL-17) has beenshown to be one of the major cytokines involved in the devel-opment and recruitment of neutrophils (18). In the presentstudy, the role of IL-17 receptor (IL-17R)-mediated signalingin the generation of protective immunity against an intracel-lular parasite was evaluated for the first time.IL-17R/ mice are more susceptible to T. gondii than areparental wild types. To determine if a lack of IL-17 signalingcan alter the susceptibility to T. gondii infection, 5- to 6-week-old female IL-17R/ mice (Amgen Inc., Thousand Oaks,Calif.) and parental C57BL/6 mice (Jackson Laboratories, BarHarbor, Maine) were challenged perorally with 15 cysts of the76K strain of T. gondii. As seen in Fig. 1A, nearly 80% of theknockout mice succumbed to infection by day 22 postinfection(p.i.). Conversely, none of the wild-type mice died or exhibitedany signs of clinical sickness, and they survived until the ter-mination of the experiment. When the inoculum was increasedto 30 cysts, parental control animals survived longer (P 0.001) but ultimately succumbed to the infection (Fig. 1B).To determine if the susceptibility of IL-17R/ mice wasdue to increased parasite burden, tissues from both knockoutand parental mice were analyzed by quantitative PCR as al-ready described (4). The relative abundance of the B1 gene, agenetic marker for T. gondii, was determined at day 14 p.i. inthe animals receiving the low dose (15 cysts) and at day 7 p.i.in those infected with a higher dose (30 cysts). As shown in Fig.1C, a low-dose infection resulted in an approximately 2 logincrease of parasite burden in the tissues (spleen, liver, gut,and brain) of knockout animals compared to wild-type mice(P  0.05). The levels in the parental controls were close touninfected levels, indicating that they were able to resolve theinfection. Similarly, when the mice were infected with a higherparasite dose, tissues of the knockout mice had a significantlyhigher parasite burden than the parental strain, with the liverdisplaying the largest difference (P  0.05) (Fig. 1D).IL-17R/ mice display less severe tissue damage in re-sponse to T. gondii infection. Previous studies have reportedthat C57BL/6 mice infected orally with a high dose of T. gondiicysts develop a severe inflammatory response (8, 15). To de-termine if lack of IL-17R can alter the inflammatory response,tissues from mice at day 7 p.i were subjected to histopatholog-ical analysis as previously described (4). As expected, the liverfrom parental C57BL/6 mice showed extensive fatty change inhepatocytes (Fig. 2A), while IL-17R-knockout mice hadslightly less severe changes (Fig. 2B). As shown in Fig. 2C, thesmall intestine of the wild-type animals exhibited extensivenecrosis and hemorrhage as previously described (10), but theIL-17R/ animals showed much less mucosal damage (Fig.2D). T. gondii tachyzoites were evident in the lamina propria ofthe IL-17R/ mice (Fig. 2, insert), while the multiple fieldsfrom the wild-type mice did not show any presence ofparasites.PMN influx into tissues is inhibited in IL-17R/ mice. Ourpresent studies demonstrated that although IL-17R/ miceexhibit increased mortality from toxoplasma infection, theydevelop a normal antigen-specific T-cell immunity and NK-cell* Corresponding author. Mailing address: Department of Microbi-ology, Immunology and Parasitology, Louisiana State UniversityHealth Sciences Center, New Orleans, LA 70112. Phone: (504) 568-6116. Fax: (504) 568-2918. E-mail: ikhan@lsuhsc.edu.617FIG.1.IL-17R/micearesusceptibletooralT.gondiiinfectionanddisplayahighparasiteburden.SurvivalofIL-17R/miceinfectedwithdifferentdosesofcysts(15[A]or30[B]cysts/mouse)isshown.Dataarerepresentedasthecumulativepercentageoftwoexperiments(n10).ThestatisticalanalysiswasperformedusingtheKaplan-Meiertest(16).ShownisthenumberofparasitespermicrogramoftissueDNAintheorgansofIL-17R/miceinfectedwith15(C)or30(D)cystsofT.gondii(n3).StatisticalanalysiswasperformedusinganunpairedStudent’sttestasalreadydescribed(12).618 NOTES INFECT. IMMUN.response against the parasite (data not shown). As neutrophilsare important in host defense to T. gondii (1, 13) and IL-17 hasbeen implicated in polymorphonuclear leukocytes (PMN)granulopoeisis and recruitment (9, 11, 18), we were interestedin evaluating the trafficking of PMNs to the tissues of animalsinfected with 30 cysts of the 76K strain of T. gondii. Hematox-ylin and eosin-stained sections of liver and intestine were ex-amined, and relative PMN indices were determined by exam-ination under an Olympus Van Ox microscope. Multiplesections of each tissue were counted in a blinded manner (7).At days 2 and 4 p.i., a decreased PMN index was observed inboth tissues in the IL-17R/ mice (Table 1). This differencewas more pronounced in the intestine, but the number ofPMNs began to increase in both tissues by day 6 p.i.Neutrophil migration into the peritoneal cavity is signifi-cantly decreased in IL-17R/ mice after T. gondii infection. Inorder to assess the ability of IL-17R/ mice to recruit neu-trophils, wild-type and knockout animals were injected intra-peritoneally with 2  106 RH strain tachyzoites. Four hourslater, peritoneal exudate cells were collected (2  105/sample)and cytospun (700 rpm for 5 min) onto glass microscope slides(Fisher Scientific, Pittsburgh, Pa.) pretreated with Vectabond(Vector Lab, Burlingame, Calif.) by using cytofunnels (ThermoShandon, Pittsburgh, Pa.). To determine the number of neu-trophils, differential counts (300 cells/slide) were performedusing a Diff-Quick stain (IMEB, San Marco, Calif.) (5). Pre-vious studies (2) have shown that intraperitoneal infection ofmice with tachyzoites of T. gondii results in a rapid recruitmentof PMNs into the peritoneal cavity. As shown in Fig. 3, thenumber of neutrophils in the peritoneum of the wild-type miceas a result of T. gondii infection was significantly higher thanthat of the IL-17R/ mice 4 h p.i. (P  0.05).To determine if a defect in neutrophil recruitment is accom-panied by decreased macrophage inflammatory protein 2(MIP-2) levels, serum from the infected animals was evaluatedfor the chemokine at days 0, 2, 4, and 6 p.i. by using anenzyme-linked immunosorbent assay kit (R&D Systems) ac-cording to the manufacturer’s protocol (14, 17). As shown inFig. 4, there was no difference between control and knockoutmice in the initial chemokine level (day 0). However, at days 2and 4 p.i., levels of MIP-2 in the control animals increasedsignificantly (P  0.02) over those in the knockout mice, whichremained at concentrations similar to those of uninfected an-imals (Fig. 4). By day 6, the level of MIP-2 in the knockoutFIG. 2. Photomicrographs of the liver and ileum from IL-17R/and wild-type mice infected with 30 cysts of T. gondii at day 7 p.i.(A) Wild-type (WT) liver: extensive fatty change is seen in hepatocyteswith small foci of mixed PMN and lymphocytic infiltration. Bar, 10 M.(B) IL17R/ liver: less severe fatty change is seen in hepatocytes withlymphocytic inflammation similar to that seen in the parental animals,including some PMNs. Bar, 10 M. (C) WT small intestine: extensivenecrosis and hemorrhage, with loss of the superficial mucosa and bloodand cellular debris in bowel lumen (star). Small foci of mixed inflam-matory cells including PMNs are seen in the lamina propria (arrow).Bar, 75 M. (D) IL 17R/ small intestine: the superficial mucosa ispreserved (star), and a mixed inflammatory infiltrate including PMNsis evident within the lamina propria (arrow). Bar, 75 M. (Inset)Close-up with arrow pointing to tachyzoites in lamina propria of aknockout animal.TABLE 1. Relative PMN indices in the tissues of T. gondii-infected miceTissuePMN indexa at day p.i.:0 2 4 6Mouse 1 Mouse 2 Mouse 1 Mouse 2 Mouse 1 Mouse 2 Mouse 1 Mouse 2Wild-type liver        IL-17R/ liver        Wild-type gut        IL-17R/ gut        a , no, or rare, single PMN within inflammatory nodules; , few, or less than one-third, of the cells in the inflammatory nodules are PMNs; , moderate, orone- to two-thirds of the cells in the nodule are PMNs; , two-thirds or more of the cells are PMNs.VOL. 73, 2005 NOTES 619mice began to increase but was still significantly less than in theparental control animals (P  0.05).In summary, as stated above, PMNs appear to play an im-portant role during early T. gondii infection (5). The mecha-nism of rapid induction of neutrophil response during T. gondiiinfection has not been described. The present study demon-strates for the first time that early neutrophil induction duringT. gondii infection is dependent on IL-17-mediated signaling.A diminished response in IL-17R/ mice was associated withfailure to produce the chemokine MIP-2 early in infection.Based on our findings, we hypothesize that T. gondii infectioninduces a strong and early neutrophil response. The neutro-phils clear the parasites during initial stages of infection so thatadaptive immunity, which is induced later, is not overbur-dened. In the presence of suboptimal levels of neutrophils,parasite load is not efficiently reduced and adaptive immunityis unable to handle this elevated burden, which ultimately leadsto increased mortality.We are thankful to Joseph Chaiban for his help in phenotypicstudies.This work was supported by National Institutes of Health grantA133325 awarded to I.A.K.REFERENCES1. Alexander, J., T. M. Scharton-Kersten, G. Yap, C. W. Roberts, F. Y. Liew,and A. Sher. 1997. 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Interleukin-17/Interleukin-17 Receptor-Mediated Signaling Is Important for Generation of an Optimal Polymorphonuclear Response against Toxoplasma gondii Infection

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Interleukin-17/Interleukin-17 Receptor-Mediated Signaling Is Important for Generation of an Optimal Polymorphonuclear Response against Toxoplasma gondii Infection

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