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IL-10 Is an Important Mediator of the Enhanced Susceptibility to Pneumococcal after

This information is current as Koenraad F. van der Sluijs, Leontine J. R. van Elden, of September 30, 2021. Monique Nijhuis, Rob Schuurman, Jennie M. Pater, Sandrine Florquin, Michel Goldman, Henk M. Jansen, René Lutter and Tom van der Poll J Immunol 2004; 172:7603-7609; ;

doi: 10.4049/jimmunol.172.12.7603 Downloaded from http://www.jimmunol.org/content/172/12/7603

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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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-10 Is an Important Mediator of the Enhanced Susceptibility to after Influenza Infection

Koenraad F. van der Sluijs,*†‡ Leontine J. R. van Elden,¶ Monique Nijhuis,¶ Rob Schuurman,¶ Jennie M. Pater,* Sandrine Florquin,¤ Michel Goldman,ʈ Henk M. Jansen,† Rene«Lutter,†‡ and Tom van der Poll1* Secondary pneumococcal pneumonia is a serious during and shortly after influenza infection. We established a mouse model to study postinfluenza pneumococcal pneumonia and evaluated the role of IL-10 in defense against pneumoniae after recovery from influenza infection. C57BL/6 mice were intranasally inoculated with 10 median tissue culture infective doses of influenza A (A/PR/8/34) or PBS (control) on day 0. By day 14 mice had regained their normal body weight and had cleared influenza from the , as determined by real-time quantitative PCR. On day 14 after viral infection, mice received 104 CFU of S. pneumoniae (serotype 3) intranasally. Mice recovered from influenza infection were highly susceptible to Downloaded from subsequent pneumococcal pneumonia, as reflected by a 100% lethality on day 3 after bacterial infection, whereas control mice showed 17% lethality on day 3 and 83% lethality on day 6 after . Furthermore, 1000-fold higher bacterial counts at 48 h after infection with S. pneumoniae and, particularly, 50-fold higher pulmonary levels of IL-10 were observed in influenza-recovered mice than in control mice. Treatment with an anti-IL-10 mAb 1 h before bacterial inoculation resulted in reduced bacterial outgrowth and markedly reduced lethality during secondary compared with those in IgG1

control mice. In conclusion, mild self-limiting influenza A infection renders normal immunocompetent mice highly susceptible to http://www.jimmunol.org/ pneumococcal pneumonia. This increased susceptibility to secondary bacterial pneumonia is at least in part caused by excessive IL-10 production and reduced function in the lungs. The Journal of Immunology, 2004, 172: 7603Ð7609.

nfluenza usually cause only mild symptoms, such which indicates that the enhanced susceptibility is not only due to as , , , sneezing, and nausea, accom- an increased viral virulence (7). Influenza infection is known to I panied by decreased activity and food intake (1). Although increase adherence of and subsequent colonization with bacterial influenza alone may lead to pneumonia, secondary bacterial infec- respiratory . may adhere to the basal membrane tions during and shortly after recovery from influenza infections after disruption of the airway epithelial layer by the cytopathic are much more common causes of pneumonia (2). The excess mor- effect of the virus (8). It has also been suggested that the increased by guest on September 30, 2021 tality rates during the of 1918Ð1919 and 1957Ð1958 adherence is due to up-regulation of receptors involved in the at- can mainly be attributed to secondary bacterial complications (3). tachment of these bacteria (9). Alternatively, influenza virus alters Even today, secondary bacterial pneumonia causes at least 20,000 the innate immune response of the host to subsequent bacterial deaths each year in the U.S. (4). Bacteria such as Staphylococcus challenges as well. Several researchers showed that influenza-in- aureus and Haemophilus influenzae are known to cause postinflu- fected mice were more sensitive to bacterial components, such as enza pneumonia, but is the most prom- staphylococcal enterotoxin B and LPS (10, 11). such as inent causing secondary bacterial pneumonia in recent IFN-␥, TNF-␣, and IL-6 are synergistically up-regulated by staph- decades (1). Primary infection with this pathogen is usually less ylococcal enterotoxin B or LPS during influenza infections in severe than secondary infection (5). mice. Influenza virus has also been reported to reduce neutrophil The severity of secondary bacterial pneumonia during or shortly activity in mice, which results in decreased pulmonary clearance after influenza infection is determined by a complex interaction after secondary bacterial infection with S. pneumoniae (11). These among virus, bacteria, and host. During combined viral/bacterial data clearly indicate that influenza virus dramatically alters the innate infections, the severity of the infection can increase due to en- immune response to bacterial infections. To date, little has been hanced virulence of the influenza virus facilitated by bacterial pro- known about the mechanism by which influenza virus modulates the teases (6). However, the host remains more susceptible to bacterial innate immune response to bacterial infections of the lungs. infections for several weeks after clearance of the influenza virus, Host defense against pneumococcal pneumonia is coordinated by the action of proinflammatory and anti-inflammatory cytokines *Laboratory of Experimental Internal Medicine, †Department of , ‡Lab- (12, 13). In the current study we present a mouse model to study oratory of Experimental Immunology, and ¤Department of Pathology, Academic host defense against S. pneumoniae after recovery from influenza ¶ Medical Center, University of Amsterdam, Amsterdam, The Netherlands; Eijkman- infection. We show that the enhanced susceptibility of mice re- Winkler Institute, Department of , University Medical Center, Utrecht, The Netherlands; and ʈDepartment of Immunology, Erasme Hospital, Universite«Libre de covered from influenza infection to secondary pneumococcal Brussels, Brussels, Belgium pneumonia is accompanied by an exaggerated production of proin- Received for publication August 29, 2003. Accepted for publication April 7, 2004. flammatory and anti-inflammatory cytokines. The finding of strik- The costs of publication of this article were defrayed in part by the payment of page ingly elevated pulmonary IL-10 concentrations in mice with charges. This article must therefore be hereby marked advertisement in accordance postinfluenza pneumonia compared with mice with primary S. with 18 U.S.C. Section 1734 solely to indicate this fact. pneumoniae pneumonia and our previous finding of a detrimental 1 Address correspondence and reprint requests to Dr. Tom van der Poll, Laboratory of Experimental Internal Medicine, Room G2-130, Academic Medical Center, Meibergdreef role for endogenous IL-10 during primary pneumococcal pneumo- 9, 1105 AZ Amsterdam, The Netherlands. E-mail address: [email protected] nia (14) prompted us to determine the contribution of exaggerated

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 7604 ROLE OF IL-10 IN POSTINFLUENZA PNEUMONIA

IL-10 production during secondary bacterial pneumonia to the en- Determination of bacterial outgrowth hanced susceptibility to S. pneumoniae of mice recovered from Serial 10-fold dilutions of the homogenates in sterile saline and 10-␮l influenza. volumes were plated out onto blood-agar plates. Plates were incubated at

37¡Cat5%CO2, and CFUs were counted after 16 h. Materials and Methods Histopathological analysis Mice Lungs for histological examination were harvested 48 h after pneumococ- Pathogen-free, 8-wk-old, female C57BL/6 mice were obtained from Harlan cal infection, fixed in 10% formalin, and embedded in paraffin. Four-mi- Sprague Dawley (Horst, The Netherlands) and maintained at biosafety cron sections were stained with H&E and analyzed by a pathologist who level 2. All experiments were approved by the animal care and use com- was blinded to the groups. mittee of Academic Medical Center, University of Amsterdam. Bronchoalveolar lavage (BAL) Experimental infection protocol The was exposed through a midline incision and cannulated with a Influenza A/PR/8/34 (ATCC VR-95; American Type Culture Collection, sterile 22-gauge Abbocath-T catheter (Abbott, Sligo, Ireland). BAL was Manassas, VA) was grown on LLC-MK2 cells (RIVM, Bilthoven, The performed by instillation of two 0.5-ml aliquots of sterile saline into the Netherlands). Virus was harvested by a freeze/thaw cycle, followed by right lung. The retrieved BAL fluid (ϳ0.8 ml) was spun at 260 ϫ g for 10 centrifugation at 680 ϫ g for 10 min. Supernatants were stored in aliquots min at 4¡C, and the pellet was resuspended in 0.5 ml of sterile PBS. Total at Ϫ80¡C. Titration was performed in LLC-MK2 cells to calculate the cell numbers were counted using a Z2 Coulter Particle Count and Size 2 median tissue culture infective dose (TCID50) of the viral stock (15). A Analyzer (Beckman Coulter, Miami, FL). Differential cell counts were per- noninfected cell culture was used for preparation of the control inoculum. formed on cytospin preparations stained with modified Giemsa stain (Diff- Downloaded from None of the stocks was contaminated by other respiratory , i.e., Quick; Baxter, McGraw Park, IL). influenza B; human parainfluenza types 1, 2, 3, 4A, and 4B; Sendai virus; RSV A and B; ; ; corona virus; and adenovirus, as Myeloperoxidase (MPO) activity measurements determined by PCR or cell culture. Viral stock and control stock were diluted just before use in PBS (pH 7.4). Mice were anesthetized by inha- MPO activity was measured as described previously (22). BAL was 5-fold lation of isoflurane (Abbott Laboratories, Kent, U.K.) and intranasally in- diluted in potassium-phosphate buffer (pH 6.0) supplemented with 0.5% oculated with 10 TCID50 of influenza (1400 viral copies) or control inoc- hexadecyl-trimethyl-ammonium bromide and 10 mM EDTA. MPO activ- ␮ http://www.jimmunol.org/ ulum in a final volume of 50 l of PBS. Pneumococcal pneumonia was ity was determined by measuring the H2O2-dependent oxidation of 3,3Ð induced 14 days after inoculation with influenza or control suspension 5,5-tetramethylbenzidine at 37¡C. The reaction was stopped by adding gla- according to previously described methods (14, 16, 17). In brief, S. pneu- cial acetic acid (0.2 M) to the reaction mixture. The amount of converted moniae serotype 3 (ATCC 6303) was cultured for 16 h at 37¡Cin5%CO2 3,3Ð5,5-tetramethylbenzidine was determined by measuring the OD at 655 in Todd Hewitt broth. This suspension was diluted 100 times in fresh nm. MPO activity is expressed in units per milliliter of BAL fluid. One unit medium and grown for5htomidlogarithmic phase. Bacteria were har- is defined as the amount of MPO required to yield one OD655 unit per vested by centrifugation at 2750 ϫ g for 10 min at 4¡C and washed twice minute. MPO activity measurements in BAL fluid represents the activation with ice-cold saline. After the second wash, the bacteria were resuspended status of polymorphonuclear cells present in the lung. in saline and diluted to a concentration of 2 ϫ 105 CFU/ml, which was verified by plating out 10-fold dilutions onto blood-agar plates. Mice were ␮ and chemokine measurements anesthetized by inhalation with isoflurane and were inoculated with 50 l by guest on September 30, 2021 of the bacterial suspension (104 CFU of S. pneumoniae). Control mice Lung homogenates were lysed with an equal volume of lysis buffer (300 4 received 10 CFU of S. pneumoniae as well. Body weight was measured mM NaCl, 30 mM Tris, 2 mM MgCl2, 2 mM CaCl2, 1% (v/v) Triton daily during the course of the first 14 days of the study (influenza vs X-100, 20 ng/ml pepstatin A, 20 ng/ml leupeptin, and 20 ng/ml aprotinin, control) and 48 h after S. pneumoniae infection. For experiments using pH 7.4) and incubated for 30 min on ice, followed by centrifugation at neutralizing IL-10 mAbs, mice were injected with JES5-2A5 (IgG1, 1 mg/ 680 ϫ g for 10 min. Supernatants were stored at Ϫ80¡C until further use. mouse) or LO-DNP (control IgG1, 1 mg/mouse) i.p. 1 h before inoculation Cytokines and chemokines in total lung lysates were measured by ELISA of the bacteria. JES5-2A5 has been used in several previous studies to according to the manufacturer’s protocol. Reagents for IL-6, IL-10, KC, inhibit IL-10 in mice in vivo (14, 18Ð20). and TNF-␣ measurements were obtained from R&D Systems (Abingdon, U.K.); IFN-␥ reagents were obtained from BD PharMingen (San Diego, CA). Determination of viral outgrowth Viral load was determined on days 4, 8, 12, and 14 after viral infection and Statistical analysis 48 h after pneumococcal infection (i.e., 16 days after viral infection) using real-time quantitative PCR as previously described (21). Mice (eight mice All data are expressed as the mean Ϯ SE unless stated otherwise. Differ- per time point) were anesthetized using 0.3 ml of fentanyl citrate (0.079 ences between groups were analyzed by Mann-Whitney U test. Survival was analyzed with Kaplan-Meier analysis using a log-rank test; p Ͻ 0.05 mg/ml), fluanisone (2.5 mg/ml), midazolam (1.25 mg/ml) in H2O (7.0 ml/kg i.p.) and were sacrificed by bleeding out the vena cava inferior. was considered to represent a statistically significant difference. Lungs were harvested and homogenized at 4¡C in 4 volumes of sterile saline using a tissue homogenizer (Biospec Products, Bartlesville, OK). Results Lung homogenates (100 ␮l) were treated with 1 ml of TRIzol reagent to extract RNA. RNA was resuspended in 10 ␮l of diethypyrocarbonate- Body weight loss after influenza infection and secondary treated water. cDNA synthesis was performed using 1 ␮l of the RNA bacterial pneumonia suspension and a random hexamer cDNA synthesis kit (Applera, Foster In previous studies influenza infection has been shown to induce City, CA). Five microliters of 25 ␮l of cDNA suspension was used for amplification in a quantitative real-time PCR (ABI PRISM 7700 Sequence loss of body weight (23). Therefore, body weight can be used as a Detector System; Applied Biosystems, Foster City, CA). The viral load marker to follow the course of influenza infections in mice. In this present in each sample was calculated using a standard curve of particle- study influenza infection led to a transient weight loss starting on counted influenza virus (virus particles were counted by electron micros- day 5 after viral exposure (Fig. 1A). Body weight reached its min- copy), included in every assay. The following primers were used: 5Ј- GGACTGCAGCTGAGACGCT-3Ј (forward), 5Ј-CATCCTGTTGTATAT imum on day 9 and returned to the baseline level on day 13 after GAGGCCCAT-3Ј (reverse), and 5Ј-CTCAGTTATTCTGCTGGTGCAC exposure. On day 14 both control mice and influenza-infected mice TTGCC-3Ј (5Ј-FAM-labeled probe). received 104 CFU of S. pneumoniae. Body weight decreased again after induction of pneumococcal pneumonia in mice recovered from influenza infection (Fig. 1A). The body weight of control

2 mice did not change ( p Ͻ 0.0001 vs mice with postinfluenza Abbreviations used in this paper: TCID50, median tissue culture infective dose; BAL, bronchoalveolar lavage; MPO, myeloperoxidase. pneumonia). The Journal of Immunology 7605

FIGURE 1. Body weight and viral load in the lungs after influenza infection in mice. The body weight (A)of virus-infected mice (Œ) and control mice (f) was mea- sured daily after intranasal inoculation (eight mice per group). Viral load was determined at several time points after influenza infection (six to eight mice per time point). Viral load is expressed as viral RNA copies per lung (B). Influenza virus was below the detection level (B.D.) on days 14 and 16 (i.e., 48 h after bacterial infec- tion) after viral infection. In control mice influenza also could not be detected (four mice per time point; data not shown).

Complete viral clearance within 14 days after influenza infection Increased bacterial outgrowth in mice recovered from influenza Viral load was determined by real-time quantitative PCR at several infection time points after viral infection to follow viral load in time. Rep- To further assess the host defense against secondary pneumococcal Downloaded from lication of the virus was observed between days 1 and 4 after virus infection, we determined bacterial outgrowth in the lungs. After infection (Fig. 1B). Viral load in the lungs peaked between days 4 48 h, just before the first deaths occurred, mice recovered from and 8 after influenza infection. On day 14 after viral infection, influenza infection showed a 1000-fold higher bacterial outgrowth influenza virus could not be detected in lung homogenates, indi- compared with mice with primary pneumococcal infection ( p ϭ cating that the virus had been cleared from the lungs. Although 0.0002 vs control mice; Fig. 3). several bacteria are known to increase the virulence of the influ- http://www.jimmunol.org/ enza virus, S. pneumoniae was not able to induce viral outgrowth Histopathological analysis on day 16 after viral infection, which confirms that the influenza Forty-eight hours after pneumococcal infection, lungs were har- virus had been completely cleared from the lungs on day 14 after vested to prepare H&E-stained lung slides for histopathological viral infection. examination. Primary pneumococcal infection resulted in intersti- tial inflammation, , endothelialitis, and pleuritis, as Increased lethality after pneumococcal infection in mice reflected by granulocyte infiltrates present in ϳ20% of the lung recovered from influenza infection (Fig. 4). Mice recovered from influenza infection with secondary Lethality was monitored at least twice a day after pneumococcal pneumococcal pneumonia showed severe interstitial inflammation, by guest on September 30, 2021 infection in mice (12 mice/group) previously infected with influ- bronchiolitis, endothelialitis, and pleuritis in the entire lung. In enza or control mice. Mice recovered from influenza infection ap- contrast to mice with primary pneumococcal infection, the infil- peared to be highly susceptible to secondary bacterial pneumonia, trates in the lungs of mice recovered from influenza consisted of as reflected by increased lethality after pneumococcal infection not only granulocytes, but also lymphocytes. These features of with 104 CFU of S. pneumoniae ( p Ͻ 0.0001 vs control mice; Fig. severe pneumonia are obviously the result of secondary infection 2). Pneumococcal pneumonia resulted in 100% lethality in mice with S. pneumoniae, as no significant pathological findings were recovered from influenza infection by day 3 and, in comparison, observed on day 12 after primary influenza virus infection (data 17% lethality in mice with primary pneumococcal infection. Le- not shown). These observations indicate that pneumococcal infec- thality in mice with primary pneumococcal infection increased up tion in mice induces more severe pulmonary inflammation after to 83% after 6 days (Fig. 2); mortality in the remaining mice did recovery from influenza infection. not occur thereafter.

FIGURE 3. Enhanced outgrowth of pneumococci during postinfluenza FIGURE 2. Previous exposure to influenza results in enhanced lethality pneumonia. Bacterial outgrowth in the lungs after pneumococcal infection due to S. pneumoniae infection. Survival after pneumococcal infection in in mice previously infected with influenza (Œ) and control mice (f). All mice previously infected with influenza (Œ) vs control mice (f). All mice mice (eight mice per group) received 104 CFU of S. pneumoniae on day 14 (12 mice/group) received 104 CFU of S. pneumoniae on day 14 after viral after viral infection and were sacrificed 48 h later. Horizontal lines repre- infection and were monitored at least twice a day after pneumococcal sent medians for each group. Note that one mouse in the control group had infection. cleared the bacteria at 48 h after infection (not shown in graph). 7606 ROLE OF IL-10 IN POSTINFLUENZA PNEUMONIA

FIGURE 4. Increased lung inflammation during postinfluenza pneumonia. Histopatho- logical analysis of the lungs of mice infected with influenza (C and D) or sham-infected control mice (A and B) was performed. All mice received 104 CFU of S. pneumoniae on day 14 after viral infection. Lungs were iso- lated 48 h after pneumococcal infection and prepared for histopathological analysis. A 10- fold magnification (A and C) was used to com- pare the area of inflamed tissue. A 100-fold magnification (B and D) was used to identify infiltrating cells in the pulmonary compart- ment. Slides are representative for six mice per group. Downloaded from

Leukocyte influx in BAL fluid estingly, mice recovered from influenza showed 20-fold higher To further assess innate immunity to S. pneumoniae after recovery concentrations of IL-10 in lung homogenates than mice not pre- viously exposed to influenza. Pulmonary cytokine levels were sim-

from influenza infection, BAL was performed to identify leukocyte http://www.jimmunol.org/ influx into the lungs. Mice recovered from influenza infection ilar in control mice and influenza-infected mice on days 14 and 16 demonstrated a substantial influx of leukocytes 48 h after infection after infection (Fig. 6). with S. pneumoniae, whereas primary pneumococcal infection as well as primary influenza infection caused only moderate recruit- Anti-IL-10 reduces bacterial outgrowth ment of leukocytes to the lungs (Table I). The increase in leuko- cyte numbers in BAL fluid after secondary pneumococcal pneu- IL-10 has been found to impair host defense during primary pneu- monia was mainly due to the recruitment of granulocytes (Table I). mococcal pneumonia (14). In light of the markedly elevated IL-10 Remarkably, mice with secondary pneumococcal pneumonia concentrations in lungs of mice with postinfluenza pneumococcal showed a significantly lower number of lymphocytes in BAL fluid pneumonia, we considered it of interest to determine the contri- by guest on September 30, 2021 than saline-treated mice recovered from primary influenza infec- bution of IL-10 in the reduced antibacterial defense of these mice. tion (Table I). Although granulocyte numbers were significantly For this, mice recovered from influenza infection were treated with higher after secondary pneumococcal pneumonia, MPO activity in a neutralizing mAb against IL-10 1 h before pneumococcal inoc- BAL fluid appeared to be similar in mice with primary and sec- ulation. Forty-eight hours after pneumococcal infection, bacterial ondary pneumococcal infections (Fig. 5). These data indicate that outgrowth was significantly lower in anti-IL-10-treated mice com- granulocyte activity is relatively reduced during secondary pneu- pared with control IgG1-treated mice ( p ϭ 0.02; Fig. 7). Thus, the mococcal pneumonia. high IL-10 levels after infection with S. pneumoniae in mice re- covered from influenza impaired bacterial clearance. Increased cytokine and chemokine levels in lung homogenates of mice recovered from influenza Pneumococcal infection elicits a number of inflammatory re- Cytokine response after treatment with anti-IL-10 sponses within the lungs. These responses include the production Cytokine levels in lung homogenates were measured to determine of proinflammatory cytokines (TNF-␣, IL-6, and IFN-␥) and che- the effect of anti-IL-10 on the induction of proinflammatory me- mokines (KC). Proinflammatory cytokine and chemokine levels diators (Fig. 8). IL-6 and KC production were similar in anti-IL- appeared to be 3- to 10-fold higher in lung homogenates of mice 10-treated mice and IgG1 control-treated mice. TNF-␣ and IFN-␥ with postinfluenza pneumococcal pneumonia compared with mice levels were lower in anti-IL-10-treated mice than in control mice with primary pneumococcal infection (all p Ͻ 0.05; Fig. 6). Inter- (both p Ͻ 0.05).

Table I. Leukocytes in BAL fluida

Cells (ϫ103) PBS ϩ Saline PBS ϩ S. pneumoniae Influenza ϩ Salineb Influenza ϩ S. pneumoniaeb,c,d

Total cell count 116 Ϯ 22 240 Ϯ 55b 315 Ϯ 43 2231 Ϯ 416 Granulocytes 2.1 Ϯ 0.7 39 Ϯ 23b 16 Ϯ 6 1734 Ϯ 340 105 Ϯ 21 199 Ϯ 39b 224 Ϯ 31 476 Ϯ 88 Lymphocytes 2.3 Ϯ 0.8 1.9 Ϯ 0.7 75 Ϯ 16 27 Ϯ 10

a Mice (n ϭ 6 per group) received PBS or influenza A intranasally at day 0, followed by S. pneumoniae intranasally or saline at day 14. BAL fluid was obtained 48 h after intranasal administration of S. pneumoniae or saline. All data are mean Ϯ SE. b p Ͻ 0.05 compared to PBS and saline-treated mice. c p Ͻ 0.05 compared to mice with primary pneumococcal infection. d p Ͻ 0.05 compared to saline-treated mice recovered from influenza infection. The Journal of Immunology 7607

FIGURE 5. MPO activity in BAL fluid. MPO activity in BAL fluid was measured for control mice, primary influenza-infected mice, primary S. pneumoniae-infected mice, and secondary S. pneumoniae-infected mice on day 16 after viral inoculation, i.e., 48 h after pneumococcal infection. Mice Downloaded from

(six mice per group) received 10 TCID50 of influenza or PBS on day 0 and 104 CFU of S. pneumoniae or saline on day 14 after viral infection. Data p Ͻ 0.05 vs control ,ء .(are expressed as units per milliliter (mean Ϯ SE mice and primary influenza-infected mice.

Anti-IL-10 improves survival during secondary pneumococcal http://www.jimmunol.org/ infection To assess whether the increased IL-10 levels adversely influenced survival during postinfluenza pneumonia, lethality was monitored in mice (12 mice/group) treated with the anti-IL-10 Ab or the IgG1 control Ab (Fig. 9). Anti-IL-10 appeared to be protective against S. pneumoniae-induced lethality in mice recovered from influenza infection, as reflected by prolonged survival and increased survival rates compared with control Ab-treated mice ( p ϭ 0.0005). by guest on September 30, 2021

Discussion Secondary bacterial infections are serious complications during and shortly after infections with influenza virus. Several mechanisms have been proposed to explain the enhanced susceptibility to secondary bacterial infections after viral infection. Many studies focused on the virus-induced damage of the respiratory FIGURE 6. Lung cytokine and chemokine concentrations on days 14 epithelium as a factor in the enhanced susceptibility to secondary and 16 after primary viral infection and 48 h after primary and postinflu- bacterial infections (8, 24Ð26). Other investigations have pointed to a enza pneumococcal pneumonia (ϩ S. pneu). Cytokine and chemokine lev- decreased cellular immune response to bacteria, reflected by reduced els in total lung homogenates were measured for mice previously infected chemotaxis and phagocytic capacity (27Ð29). We show in this study with influenza virus (Ⅺ) and control mice (f). Mice (eight mice per group) that pneumococcal pneumonia in mice recovered from influenza in- received 104 CFU of S. pneumoniae on day 14 after viral infection. Pul- fection is associated with profoundly elevated IL-10 concentrations in monary levels of TNF-␣, IFN-␥, IL-6, KC, and IL-10 are expressed as p Ͻ 0.05 vs control ,ء .(the lungs, and that these IL-10 levels contribute to the increased sus- nanograms per gram of lung tissue (mean Ϯ SE ceptibility to secondary bacterial pneumonia. mice with primary pneumococcal pneumonia. Our present finding that influenza virus infection renders mice more susceptible to pneumonia caused by S. pneumoniae confirms earlier reports of bacterial complications following influenza infection infection from influenza virus. Indeed, mice with postinfluenza pneu- (9, 11). In the earlier investigations mice were infected with S. pneu- mococcal pneumonia showed an increased lethality and an enhanced moniae on day 7 after inoculation with influenza virus. Our model for bacterial outgrowth in the lungs compared with mice with primary secondary bacterial pneumonia was designed to exclude direct inter- pneumococcal pneumonia. actions between influenza virus and S. pneumoniae. In particular, Mice with S. pneumoniae pneumonia preceded by influenza in- mice received the bacterial inoculum on day 14 after viral exposure, fection displayed an enhanced inflammatory response compared i.e., at the point the virus was completely cleared from the lungs. By with mice with primary pneumococcal infection, as indicated by then, these mice had clinically recovered from influenza infection, as histopathology, cell influx in BAL fluid, and higher cytokine and reflected by normal bodyweight. This time point was also chosen chemokine levels in lung homogenates. Probably this exaggerated because clinical data indicate that 2 wk is a common interval between inflammation was the consequence of the much higher bacterial influenza infection and the occurrence of secondary bacterial compli- load, providing a stronger proinflammatory stimulus in these cations (1, 2). Our data clearly indicate that mice remain highly sus- postinfluenza mice. In support of this idea are previous findings by ceptible to secondary bacterial infections after recovery from airway Dallaire et al. (30), demonstrating that pulmonary cytokine levels 7608 ROLE OF IL-10 IN POSTINFLUENZA PNEUMONIA

FIGURE 7. Anti-IL-10 reduces bacterial outgrowth during postinflu- enza pneumonia. S. pneumoniae CFU in the lungs after pneumococcal FIGURE 9. Anti-IL-10 improves survival during postinfluenza pneu- Œ infection in mice treated with a neutralizing Ab against IL-10 ( ) and monia. Survival after pneumococcal infection in mice treated with a neu- f control IgG1-treated mice ( ). All mice (eight mice per group) received 10 tralizing Ab against IL-10 (Œ) vs control IgG1-treated mice (f). Mice (12 TCID of influenza on day 0 and 104 CFU of S. pneumoniae on day 14 4 50 mice/group) received 10 TCID50 of influenza on day 0 and 10 CFU of S. after viral infection and were sacrificed 48 h after pneumococcal infection. pneumoniae on day 14 after viral infection and were monitored at least Anti-IL-10 or control IgG1 was given i.p. 1 h before infection with S. twice a day after pneumococcal infection. pneumoniae. Horizontal lines represent medians for each group.

and postinfluenza bacterial pneumonia (despite much higher neu- correlate closely with the extent of bacterial outgrowth during trophil numbers in the latter group). Similar results were obtained Downloaded from pneumococcal pneumonia. Alternatively, colonization of S. pneu- by LeVine et al., who showed that MPO release by isolated bron- moniae may be due to the damage of the epithelial layer caused by choalveolar lavage was significantly lower after pneu- an exaggerated inflammatory response. However, our data do not mococcal infection in mice previously exposed to influenza (Ϫ7 support this latter explanation, as neutralization of IL-10, an anti- days) than in control mice (11). Considering that IL-10 potently inflammatory cytokine, appears to be protective. inhibits neutrophil functions, including degranulation (31Ð33), we

Mice recovered from influenza infection displayed much higher consider it highly likely that the elevated IL-10 levels in mice with http://www.jimmunol.org/ bacterial counts after infection with S. pneumoniae, yet had 50- postinfluenza pneumonia are at least in part responsible for the fold higher neutrophil numbers in BAL fluid compared with mice relatively reduced neutrophil function. with primary pneumococcal pneumonia. Probably the increased In primary S. pneumoniae infection of the airways, endog- neutrophil numbers were the result of the higher bacterial load, enously produced IL-10 impairs host defense (14). Similarly, providing a more potent proinflammatory stimulus. Indeed, in mu- IL-10 hampered an adequate immune response during murine rine pneumococcal pneumonia the extent of inflammation is pneumonia (34). These data led us to hy- closely correlated with the number of bacteria (30). Moreover, our pothesize that the strongly elevated pulmonary levels of IL-10 in data indicate that granulocyte function was significantly reduced mice with postinfluenza pneumococcal pneumonia were important during secondary pneumococcal pneumonia, as reflected by sim- for the impairment of defense against S. pneumoniae. Our current by guest on September 30, 2021 ilar MPO activities in BAL fluid obtained in mice with primary findings provide evidence for this hypothesis, i.e., anti-IL-10 treat- ment directly before infection with S. pneumoniae reduced the outgrowth of pneumococci in lungs and increased survival, sug- gesting a direct link between elevated IL-10 levels and enhanced susceptibility to secondary bacterial infection. Interestingly, Stein- hauser et al. (35) used an approach identical with that taken in the current study to establish that enhanced IL-10 production contrib- uted significantly to the diminished lung antibacterial defense against aeruginosa in mice with abdominal induced by cecal ligation and puncture. Together these data point to an immunosuppressive effect of endogenous IL-10 in the respi- ratory tract for highly different conditions (abdominal sepsis and influenza infection of the airways). Proinflammatory mediators such as IL-6 and KC were not in- fluenced by anti-IL-10 treatment, whereas TNF-␣ and IFN-␥ con- centrations even were decreased in anti-IL-10-treated mice. These findings contrast with earlier findings in primary pneumococcal pneumonia, where anti-IL-10 treatment resulted in increased TNF-␣ and IFN-␥ concentrations in the lungs (14). Although IL-10 is expected to inhibit the production of proinflammatory cytokines, it is conceivable that the reduced bacterial load in the lungs of mice administered anti-IL-10 resulted in an attenuated FIGURE 8. Effect of anti-IL-10 on cytokine and chemokine concentra- proinflammatory stimulus and masked the potentiating effect of tions in the lung. Cytokine and chemokine levels in total lung homogenates anti-IL-10 on the production of these mediators. In line with this, were measured in mice treated with a neutralizing Ab against IL-10 (Ⅺ) Dallaire et al. (30) have shown that proinflammatory cytokine lev- and control IgG1-treated mice (f). All mice (eight mice per group) re- 4 els in lungs of mice with pneumococcal pneumonia closely corre- ceived 10 TCID50 of influenza on day 0 and 10 CFU of S. pneumoniae on day 14 after viral infection. TNF-␣ (upper left graph), IFN-␥ (lower left late with the bacterial load. Likewise, in a model of mono- graph), IL-6 (upper right graph), and KC (lower right graph) were mea- cytogenes infection, anti-IL-10 treatment reduced bacterial sured. Data are expressed as nanograms per gram of lung tissue (mean Ϯ outgrowth and concurrently inhibited the expression of proinflam- .(p Ͻ 0.05 vs IgG1-treated mice. matory cytokine in the liver (36 ,ء .(SE The Journal of Immunology 7609

Of note, some difference existed in the mortality curves of mice 9. McCullers, J. A., and J. E. Rehg. 2002. Lethal synergism between influenza virus with postinfluenza pneumococcal pneumonia that were not treated and Streptococcus pneumoniae: characterization of a mouse model and the role of platelet-activating factor receptor. J. Infect. Dis. 186:341. with anti-IL-10, as shown in Figs. 2 and 9. Although only in the latter 10. Zhang, W. J., S. Sarawar, P. Nguyen, K. Daly, J. E. Rehg, P. C. Doherty, experiment were mice treated with control IgG, we consider it likely D. L. Woodland, and M. A. Blackman. 1996. Lethal synergism between influenza and staphylococcal enterotoxin B in mice. J. Immunol. 157:5049. that this difference is the result of biovariability related to the fact that 11. Le Vine, A. M., V. Koenigsknecht, and J. M. Stark. 2001. Decreased pulmonary these experiments were performed at an interval of several months clearance of S. pneumoniae following influenza A infection in mice. J. Virol. using different “shipments” of mice. In addition, the model described Methods. 94:173. 12. Schultz, M. J., S. Knapp, and T. van der Poll. 2002. Regulatory role of alveolar in this manuscript uses two subsequent infections, and both infections, macrophages and cytokines in pulmonary host defense. In: Yearbook of Intensive without doubt, have a certain variability in inoculum size and infec- Care and Emergency Medicine. J. L. Vincent, eds. Springer-Verlag, Berlin, p. 65 tion efficiency, leading to subtle differences in pathology. However, 13. Strieter, R. M., J. A. Belperio, and M. P. Keane. 2003. Host innate defenses in the lung: the role of cytokines. Curr. Opin. Infect. Dis. 16:193. we emphasize that all experiments were performed in an appropriately 14. van der Poll, T., A. Marchant, C. V. Keogh, M. Goldman, and S. F. Lowry. 1996. controlled way, i.e., the groups to be compared were always infected Interleukin-10 impairs host defense in murine pneumococcal pneumonia. J. In- fect. Dis. 174:994. at the same time using exactly the same inoculum. 15. Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty percent Primary influenza virus infection leads to recruitment of lym- endpoints. Am. J. Hyg. 27:s493. phocytes and macrophages, which are both able to release IL-10 16. Lauw, F. N., J. Branger, S. Florquin, P. Speelman, S. J. van Deventer, S. Akira, and T. van der Poll. 2002. IL-18 improves the early antimicrobial host response (32). Macrophages, at least in part, may be responsible for the to pneumococcal pneumonia. J. Immunol. 168:372. exaggerated IL-10 production, as numbers were further 17. Rijneveld, A. W., S. Florquin, J. Branger, P. Speelman, S. J. van Deventer, and ␣ increased after secondary infection with S. pneumoniae, whereas lym- T. van der Poll. 2001. TNF- compensates for the impaired host defense of IL-1 type I receptor-deficient mice during pneumococcal pneumonia. J. Immunol. phocyte numbers were significantly reduced. However, a 2-fold in- 167:5240. Downloaded from crease in macrophage numbers cannot fully account for a 50-fold 18. Jovasevic, V. M., L. Gorelik, J. A. Bluestone, and M. B. Mokyr. 2004. Impor- tance of IL-10 for CTLA-4-mediated inhibition of tumor-eradicating immunity. increase in IL-10 production, unless these macrophages have become J. Immunol. 172:1449. hyper-responsive to secondary bacterial challenges. An alternative ex- 19. van der Poll, T., A. Marchant, W. A. Buurman, L. Berman, C. V. Keogh, planation for this discrepancy is that other cell types produce high D. D. Lazarus, L. Nguyen, M. Goldman, L. L. Moldawer, and S. F. Lowry. 1995. Endogenous IL-10 protects mice from death during septic peritonitis. J. Immunol. amounts of IL-10 as well. In this context, lymphocytes cannot be 155:5397. excluded as a prominent source of IL-10, as lymphocyte numbers are 20. Marchant, A., C. Bruyns, P. Vandenabeele, M. Ducarme, C. Gerard, A. Delvaux, http://www.jimmunol.org/ still higher after secondary infection with S. pneumoniae than after D. De Groote, D. Abramowicz, T. Velu, and M. Goldman. 1994. Interleukin-10 controls -␥ and tumor necrosis factor production during experimental primary infection with S. pneumoniae. Further research is required to endotoxemia. Eur. J. Immunol. 24:1167. identify the cellular source of IL-10 and to unravel the mechanism by 21. Van Elden, L. J. R., M. Nijhuis, P. Schipper, R. Schuurman, and A. M. van Loon. 2001. Simultaneous detection of influenza virus A and B using real-time quan- which influenza virus predisposes to an exaggerated IL-10 production titative PCR. J. Clin. Microbiol. 39:196. upon infection with S. pneumoniae. 22. Knapp, S., J. C. Leemans, S. Florquin, J. Branger, N. A. Maris, J. 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