Interleukin-10 Produced by Myeloid-Derived Suppressor CellsProvides Protection to Carbapenem-Resistant Klebsiellapneumoniae Sequence Type 258 by Enhancing Its Clearance inthe Airways

Hernán F. Peñaloza,a* Loreani P. Noguera,a Danielle Ahn,b Omar P. Vallejos,a Raquel M. Castellanos,c Yaneisi Vazquez,a

Francisco J. Salazar-Echegarai,a Liliana González,a Isidora Suazo,a Catalina Pardo-Roa,a Geraldyne A. Salazar,a Alice Prince,b,d

Susan M. Buenoa

aMillennium Institute on Immunology and Immunotherapy, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, PontificiaUniversidad Católica de Chile, Santiago, Chile

bDepartment of Pediatrics, Columbia University Medical Center, New York, New York, USAcDepartamento de Morfología, Facultad de Medicina, Universidad Andres Bello, Santiago, ChiledDepartment of Pharmacology, Columbia University Medical Center, New York, New York, USA

ABSTRACT Carbapenem-resistant Klebsiella pneumoniae sequence type 258 (CRKP-ST258) can cause chronic infections in lungs and airways, with repeated episodes ofbacteremia. In this report we addressed whether the recruitment of myeloid cellsproducing the anti-inflammatory cytokine interleukin-10 (IL-10) modulates the clear-ance of CKRP-ST258 in the lungs and establishes bacterial persistence. Our datademonstrate that during pneumonia caused by a clinical isolate of CRKP-ST258(KP35) there is an early recruitment of monocyte-myeloid-derived suppressor cells(M-MDSCs) and neutrophils that actively produce IL-10. However, M-MDSCs were thecells that sustained the production of IL-10 over the time of infection evaluated. Us-ing mice unable to produce IL-10 (IL-10�/�), we observed that the production ofthis cytokine during the infection caused by KP35 is important to control bacterialburden, to prevent lung damage, to modulate cytokine production, and to improvehost survival. Importantly, intranasal transfer of bone marrow-derived M-MDSCs frommice able to produce IL-10 at 1 day prior to infection improved the ability of IL-10�/� mice to clear KP35 in the lungs, decreasing their mortality. Altogether, ourdata demonstrate that IL-10 produced by M-MDSCs is required for bacterial clear-ance, reduction of lung tissue damage, and host survival during KP35 pneumonia.

KEYWORDS interleukin-10, Klebsiella pneumoniae ST258, monocytic-myeloid-derivedsuppressor cells, neutrophils

One major cause of pneumonia in health care facilities worldwide is carbapenem-resistant Klebsiella pneumoniae sequence type 258 (CRKP-ST258). CRKP-ST258

strains encode KPC �-lactamases, which makes these bacteria resistant to different�-lactams, including carbapenems (1, 2). CRKP-ST258 isolates are the etiologic agent ofdifferent diseases, including pneumonia, sepsis, and urinary tract infections (3). CRKP-ST258 pneumonia in health care facilities is associated with high mortality rates (50%)although in some cases these bacteria can also establish chronic infections withfrequent episodes of sepsis (4).

Similar to the well-studied laboratory reference strain K. pneumoniae ATCC 43816(KPPR1), CRKP-ST258 expresses the typical pathogen-associated molecular patterns(PAMPs) of Gram-negative bacteria (3) that potently activate proinflammatory signalingon epithelial and immune cells through Toll-like receptor 4 (TLR4)/MyD88/NF-�B (4).

Citation Peñaloza HF, Noguera LP, Ahn D,Vallejos OP, Castellanos RM, Vazquez Y, Salazar-Echegarai FJ, González L, Suazo I, Pardo-Roa C,Salazar GA, Prince A, Bueno SM. 2019.Interleukin-10 produced by myeloid-derivedsuppressor cells provides protection tocarbapenem-resistant Klebsiella pneumoniaesequence type 258 by enhancing its clearancein the airways. Infect Immun 87:e00665-18.https://doi.org/10.1128/IAI.00665-18.

Editor Vincent B. Young, University ofMichigan—Ann Arbor

Copyright © 2019 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Susan M. Bueno,sbueno@bio.puc.cl.

* Present address: Hernán F. Peñaloza, Divisionof Pulmonary, Allergy and Critical CareMedicine, Department of Medicine, Universityof Pittsburgh, Pittsburgh, Pennsylvania, USA.

Received 27 August 2018Returned for modification 28 September2018Accepted 17 February 2019

Accepted manuscript posted online 25February 2019Published

HOST RESPONSE AND INFLAMMATION

crossm

May 2019 Volume 87 Issue 5 e00665-18 iai.asm.org 1Infection and Immunity

23 April 2019

on October 12, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

Therefore, we believe that the ability of CRKP-ST258 to cause chronic infections is notbecause of impaired bacterial recognition by the immune system. In contrast to KPPR1,CRKP-ST258 isolates have acquired resistance to killing by neutrophils (4, 5). DuringCRKP-ST258 infection, the initial neutrophil response is rapidly supplanted by therecruitment of suppressive monocytic myeloid-derived suppressor cells (M-MDSCs) (4),which have been described as major suppressors of the inflammatory response duringcancer and infection (6).

During KPPR1 pneumonia, MDSCs are recruited at later time points, and theirfunction is associated with lung recovery and remodeling after infection (7). MDSCs area heterogeneous group of anti-inflammatory cells best described in cancer (8). Thisgroup of myeloid cells is composed of at least three different subsets: monocytic(M-MDSCs), granulocytic (G-MDSCs) (9), and eosinophilic (Eo-MDSCs) cells (10). Eachsubset shares the same surface markers of their proinflammatory counterparts (inflam-matory monocytes, neutrophils, and eosinophils, respectively) but also expresses anti-inflammatory mediators able to inhibit T cell function and proliferation, such asarginase-1, inducible nitric oxide synthase (iNOS), and interleukin-10 (IL-10) (9).

IL-10 is a major anti-inflammatory cytokine produced by nearly all leukocytes, withwidely suppressive effects on several immune cell types (11). During bacterial infec-tions, IL-10 modulates the proinflammatory immune response, allowing clearance ofthe pathogen with reduced tissue damage (12). In the case of IL-10 overproduction, theimmune response is not robust enough to clear the infective pathogen, leading tochronic infections or elevated host mortality (13, 14). In this report we evaluatedwhether the early recruitment of IL-10-producing M-MDSCs observed during pneumo-nia caused by a clinical isolate of CRKP-ST258 (KP35) (4, 15) plays a major role in theestablishment of a persistent infection and delayed bacterial clearance. Using trans-genic mice expressing IL-10 and enhanced green fluorescent protein (eGFP) (IL-10/GFPmice), we demonstrate that M-MDSCs recruited early to the lungs of KP35-infected miceare the main myeloid source of IL-10 in the lungs from 48 to 240 h postinfection. Thecentral role of this myeloid population was shown by the attenuation of pulmonarydamage by the adoptive transfer into IL-10�/� mice of MDSCs able to produce IL-10.Altogether, IL-10 production in response to CRKP-ST258 infection has an importantfunction in enhancing bacterial clearance from the airways.

RESULTSNeutrophils and M-MDSCs account for the early production of IL-10 in lungs of

KP35-infected mice. To evaluate the ability of M-MDSCs to produce IL-10 at differenttimes post-KP35 infection, reporter IL-10/GFP VertX mice were intranasally inoculatedwith KP35. IL-10-producing cells were then quantified by flow cytometry. Similar towild-type (WT) mice (4), IL-10/GFP mice displayed severe symptomatology after KP35infection, with a peak symptom score at 48 h postinfection (hpi), a survival rate of 100%,a reduction in the epithelial/endothelial barrier function, and elevated bacterial burdenin the lung tissue and bronchoalveolar lavage fluid (BALF) at 10 days postinfection (dpi)(see Fig. S1 in the supplemental material). The infection was characterized by a rapidrecruitment of neutrophils and M-MDSCs, a decrease in the number of alveolar mac-rophages, and a mild recruitment of Ly6C� monocytes, eosinophils, and interstitialmacrophages (Fig. 1 and Fig. S2). As expected, several innate immune cells producedIL-10 in response to KP35. IL-10 production started at 24 hpi, with the arrival ofneutrophils, identified as the most abundant IL-10-producing cells during the first 48hpi (Fig. 1A). From 48 hpi onward, the population of IL-10-producing neutrophilsreturned to baseline levels, and recruited M-MDSCs actively produced IL-10 until 240hpi (Fig. 1B). Ly6C� monocytes were also an important source of IL-10 at 48 and 72 hpi(Fig. 1C). Moreover, eosinophils were identified as IL-10-producing cells at 72 and 96 hpi(Fig. S2A). Alveolar macrophages and interstitial macrophages did not show a signifi-cant increase in IL-10 production (Fig. S2B and C).

IL-10 production during KP35 infection is critical for host survival and bacterialclearance. Next, we evaluated whether the production of IL-10 is required for the

Peñaloza et al. Infection and Immunity

May 2019 Volume 87 Issue 5 e00665-18 iai.asm.org 2

on October 12, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

clearance of KP35 from the lungs. To address this aim, WT and IL-10 knockout (IL-10�/�)mice were intranasally infected with KP35, and bacterial burden was evaluated at 24and 48 hpi. At 48 hpi, IL-10�/� mice had elevated bacterial counts in the lung tissueand BALF but not in the spleen (Fig. 2A). IL-10�/� mice also had increased mortality(Fig. 2B), with 81% (9/11) of WT mice surviving after KP35 infection up to 7 dayswhereas only 10% (1/10) of IL-10�/� mice survived. Consistently, IL-10�/� mice also

FIG 1 Neutrophils, M-MDSCs, and Ly6C� monocytes are the main IL-10 producers during KP35 infection. (A to C)IL-10/GFP VertX mice were intranasally infected with 1 � 108 CFU of KP35, and IL-10 production by myeloid cellswas evaluated by flow cytometry at 12, 24, 48, 72, 96, and 240 hpi on neutrophils, M-MDSCs, and Ly6C� monocytes,as indicated. For each cell population, data are presented as the number of total lung cells, the numbers of lungGFP� (IL-10�) cells, the percentage of GFP� (IL-10�) cells relative to the total number of cells, and the fold changein mean fluorescence intensity (MFI) of GFP in the total cell population. WT uninfected mice were included toestablish the autofluorescence level (data not shown). *, P � 0.05, for comparison of each group with theuninfected control (by one-way ANOVA with a Tukey posttest). FITC, fluorescein isothiocyanate.

IL-10 from M-MDSCs in K. pneumoniae ST258 Pneumonia Infection and Immunity

May 2019 Volume 87 Issue 5 e00665-18 iai.asm.org 3

on October 12, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

had more severe symptomatology than WT mice (Fig. 2B and C). At 24 hpi, IL-10�/�

mice presented increased lung inflammation (Fig. 3A to C) and terminal deoxy-nucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) (Fig. 3D and E).At 48 hpi, lung inflammation remained higher in IL-10�/� mice (Fig. 3A to C), but thesemice presented a decrease in TUNEL-positive area compared to that of WT mice, whichpresented an increasing trend over time (Fig. 3D and E). Consistently, IL-10�/� mice hadreduced myeloperoxidase (MPO) activity in BALF (Fig. 3F). Despite this, IL-10�/� micepresented an elevated lung injury, evidenced in a higher histopathology score and totalBALF protein amount. Interestingly, the increased lung injury observed in IL-10�/� micedid not correlate with increased infiltration to the airways of inflammatory myeloidcells, such as neutrophils, M-MDSCs, eosinophils, and macrophages, which remained atlevels between those of IL-10�/� and WT mice at 24 and 48 h after KP35 infection (Fig.4 and Fig. S3).

IL-10 production has a major effect on proinflammatory cytokine produc-tion. Proinflammatory cytokine production was measured in BALF of infected mice.WT mice produced reduced amounts of tumor necrosis factor alpha (TNF-�), IL-1�,and IL-6 at 24 and 48 hpi (Fig. 5A to C). Furthermore, negligible amounts ofIL-12p40, IL-23p19, and gamma interferon (IFN-�) were detected in these mice (Fig.5D to F). In contrast, elevated levels of TNF-� and IL-6 were detected in BALFsamples of IL-10�/� mice at 24 hpi, and levels were even higher at 48 hpi (Fig. 5Aand B). Moreover, increased amounts of IL-1�, IL-12p40, IL-23p19, and IFN-� weredetected at 48 hpi (Fig. 5C to F).

IL-10 provided by MDSCs is important for KP35 clearance, prevention of lunginjury, and host survival. MDSCs from bone marrow of IL-10/GFP mice (BM-MDSCs)were treated and differentiated with granulocyte colony-stimulating factor (G-CSF) andgranulocyte-macrophage colony-stimulating factor (GM-CSF) over 5 days and then

FIG 2 IL-10 production is required for KP35 clearance and host survival. WT and IL-10�/� mice were intranasallyinfected with 1 � 108 CFU of KP35. (A) Bacterial burden was evaluated in lung, BALF, and spleen at 24 and 48 hpi.(B to D) Survival rate, severity of the disease, and body weight change were evaluated over 7 days († representsthe number of dead mice at 4 dpi). For bacterial burden, * P � 0.05 (Mann-Whitney U test comparing resultsbetween both groups at each time point); for the survival curve, ***, P � 0.0005 (log rank test); for disease severity(clinical score), *, P � 0.05 (two-way ANOVA with a Tukey posttest comparing results for both groups at each timepoint). ns, not significant.

Peñaloza et al. Infection and Immunity

May 2019 Volume 87 Issue 5 e00665-18 iai.asm.org 4

on October 12, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

intranasally transferred into IL-10�/� mice. Published data demonstrate that these cellsexpress the typical MDSC markers, are not able to clear bacteria, and suppress T cell andneutrophil function (4, 16, 17). Consistently in this report, differentiated cells expressedthe characteristic surface markers of MDSCs (CD11b, Ly6C, Ly6G, F4/80, CD115, majorhistocompatibility complex class II [MHC-II], and CD11c) and were not able to kill KP35in vitro (Fig. S4). Intranasal transfer of BM-MDSCs able to produce IL-10 in IL-10�/� miceat 1 day prior to infection improved KP35 clearance capacity in the lung tissue (Fig. 6A)but not in BALF (Fig. 6B) or spleen (Fig. 6C). Even though total IL-10 production (inpicograms/milliliter) was not detected in BALF of IL-10�/� mice with or withoutBM-MDSC transfer, we specifically detected the production of IL-10 by transferredIL-10/GFP-expressing BM-MDSCs (IL-10/GFP BM-MDSCs) in the lungs of IL-10�/� miceby flow cytometry (Fig. 6D to F). Production of IL-10, however, was not enough to

FIG 3 IL-10 production modulates lung damage during KP35 infection. WT and IL-10�/� mice were intranasally infected with1 � 108 CFU of KP35, and different parameters of lung damage were evaluated, including lung histopathology by H&E staining (A),lung histopathological score (B), total BALF proteins (C) and total lung cell apoptosis by TUNEL assay (D) and TUNEL-positive area(E). (F) Neutrophil activation was measured by the quantification of MPO activity in the BALF. Scale bar, 2 mm. *, P � 0.05, for acomparison of results between groups at each time point or between the infected group and the respective uninfected (Unf)group (two-way ANOVA with a Tukey posttest).

IL-10 from M-MDSCs in K. pneumoniae ST258 Pneumonia Infection and Immunity

May 2019 Volume 87 Issue 5 e00665-18 iai.asm.org 5

on October 12, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

modulate the production of proinflammatory cytokines, which remained higher in allIL-10�/� groups than in control mice without transferred IL-10/GFP (Fig. 6G).

IL-10 production by transferred IL-10/GFP BM-MDSCs not only improved KP35clearance but also slightly reduced lung injury. In fact, IL-10�/� mice that receivedIL-10/GFP BM-MDSCs presented a lung histopathological score equivalent to that ofmice that did not receive IL-10/GFP as well as equivalent amounts of total BALFproteins (Fig. 7).

When IL-10�/� mice received IL-10/GFP BM-MDSCs, they showed increased survival(P � 0.05) (Fig. 8A) and reduced symptomatology (Fig. 8B and C) (P � 0.05), which was

FIG 4 IL-10 production does not influence immune cell recruitment to the airways of KP35-infected mice.Recruitment of neutrophils (A), M-MDSCs (B), and Ly6C� (C) monocytes was measured in the lung and BALF ofuninfected and infected WT and IL-10�/� mice at 24 and 48 hpi. *, P � 0.05, for a comparison of results betweengroups at each time point or between the infected group and the respective uninfected (Unf) group (two-wayANOVA with a Tukey posttest).

FIG 5 IL-10 modulates proinflammatory cytokine production during KP35 pneumonia. (A to F) The production ofTNF-�, IL-6, IL-1�, IL-12p40, IL-23p19, and IFN-� in BALF, as indicated, from WT and IL-10�/� mice was analyzed byLuminex technology at 24 and 48 hpi. *, P � 0.05, for a comparison of results of both groups at each time point(two-way ANOVA with a Tukey posttest).

Peñaloza et al. Infection and Immunity

May 2019 Volume 87 Issue 5 e00665-18 iai.asm.org 6

on October 12, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

equivalent to levels in untransferred IL-10/GFP mice (Fig. 8). Interestingly, adoptivetransfer of IL-10�/� mouse BM-MDSCs slightly increased host survival from 0% to 25%although these mice still presented an elevated clinical score (Fig. 8). Adoptive transferof BM-MDSCs from IL-10/GFP or IL-10�/� mice to IL-10/GFP-recipient mice did not

FIG 6 IL-10 produced by BM-MDSCs is critical for restoring bactericidal capacity in IL-10�/� mice. BM-MDSCs from IL-10�/� andIL-10/GFP VertX mice were generated in vitro. One day before the infection (day �1), 1 � 106 BM-MDSCs were transferredintranasally into IL-10�/� mice. At day 0 each group was intranasally infected with 1 � 108 CFU of KP35, and at 48 h postinfection,mice were euthanized. (A to C) Bacterial burden in lung tissue, spleen, and BALF was determined. (D and E) IL-10 production byMDSCs was identified by flow cytometry in the lung tissue. (F and G) Cytokine production in BALF was quantified by Luminexassay. *, P � 0.05, for a comparison of results for each parameter (by one-way ANOVA with a Tukey posttest). UT, untransferred.

IL-10 from M-MDSCs in K. pneumoniae ST258 Pneumonia Infection and Immunity

May 2019 Volume 87 Issue 5 e00665-18 iai.asm.org 7

on October 12, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

influence the resistance observed in these mice (data not shown). Cellular infiltrationwas not affected with the exception of reduced neutrophils in the total lung homog-enate (Fig. S5A) but not in the BALF (Fig. S5B).

DISCUSSION

CRKP-ST258 is a major health concern worldwide (2). Available data describe majordifferences between CRKP-ST258 and KPPR1 strains in terms of pathogenesis, virulence,and clinical manifestations (Fig. 9A and B) (4, 18, 19). In terms of virulence, the 50%lethal dose (LD50) of KPPR1 in mice is between 3 � 103 and 1.8 � 104 CFU in wild-typemice (19, 20), in contrast to levels for CRKP-ST258 isolates, for which the LD50 is muchhigher, typically around 1 � 108 CFU (4, 18). Moreover, CRKP-ST258 can establishchronic and persistent infections (4). Under these conditions, CRKP-ST258 colonizationallows adaptation to environmental conditions, such as antibiotic treatment, as well asto the host defenses (4, 5).

FIG 7 IL-10 produced by BM-MDSCs modulates lung injury during KP35 pneumonia. BM-MDSCs fromIL-10�/� and IL-10/GFP VertX mice were generated in vitro. One day before the infection (day �1),1 � 106 BM-MDSCs were transferred intranasally into IL-10�/� mice. At day 0 each group was intranasallyinfected with 1 � 108 CFU of KP35; at 48 h postinfection, mice were euthanized and lung histopathologywas assessed through H&E staining (A). Lung injury was quantified through a histopathological score (B)and through the quantification of total proteins in the BALF (C). *, P � 0.05; ns, not significant, for acomparison of results for each parameter (by one-way ANOVA with a Tukey posttest).

Peñaloza et al. Infection and Immunity

May 2019 Volume 87 Issue 5 e00665-18 iai.asm.org 8

on October 12, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

One of the most remarkable characteristics of CRKP-ST258 murine pneumonia is thenature of the immune response that is evoked, characterized by reduced production ofproinflammatory cytokines despite robust recruitment of neutrophils during the first 24hpi (4). This attenuated inflammatory response correlates with elevated levels of IL-10in the BALF (4). In the present report, we have demonstrated that during KP35 infection,the production of IL-10 by the early recruited M-MDSCs directs the immune responseand is critical for host survival. Specifically, the production of IL-10 prevents uncon-trolled cytokine production, reduces lung damage, and improves bacterial clearancealthough this comes at the expense of a delayed bacterial clearance in the airways.

IL-10 is a major anti-inflammatory cytokine that modulates excessive lung inflam-mation during infection (21). Lack of IL-10 during acute pulmonary infections typicallyleads to more severe disease, characterized by increased proinflammatory cytokineproduction, increased recruitment/activity of neutrophils and, in some cases, increasedmortality (7, 21). Whether lack of IL-10 increases or decreases host susceptibilitydepends on the specific pathogen. For example, during Streptococcus pneumoniaepneumonia, a high infective dose unmasks a critical role of IL-10 that favors hostsurvival (21). However, during KPPR1 infection, IL-10 neutralization led to improvedhost survival (Fig. 9C) (22). The data provided in this report are consistent with previousreports on the role of IL-10 in lung immunity but also highlight major differences fromwhat has been described in classical KPPR1 pneumonia (7, 22). During a sublethalinfection of KPPR1, the lack of IL-10 enhances neutrophil activity and bacterial clearancealthough it also leads to elevated cytokine production, lung damage, and more severepathology (Fig. 9C) (7). Similarly, lack of IL-10 during KP35 pneumonia also led toincreased production of proinflammatory cytokines and greater lung damage; however,we observed elevated bacterial burden in the airways, impaired neutrophil activation,and increased host mortality during KP35 infection (Fig. 9D). This highlights importantdifferences between CRKP-ST258 and KPPR1 in the nature of the innate immuneresponses that are activated in response to these common pathogens (Fig. 9A and B)and also unmasks important differences in the roles of IL-10 between CRKP-ST258 andKPPR1 pneumonia (Fig. 9C and D).

The kinetics of IL-10 production and the cellular source of IL-10 are also majordifferences between pneumonia caused by CRKP-ST258 and that caused by otherpathogens. IL-10 production is induced in myeloid cells after PAMP recognition bypattern recognition receptors (PRRs) (11). In this scenario, any leukocyte may potentiallyproduce IL-10. During S. pneumoniae pneumonia, macrophages and T cells have been

FIG 8 IL-10 produced by BM-MDSCs is critical for host survival during KP35 pneumonia. (A to C) BM-MDSCs from IL-10�/� and IL-10/GFPVertX mice were generated in vitro. One day before the infection (day �1), 1 � 106 BM-MDSCs were transferred (T) intranasally intoreceptor IL-10�/� mice. At day 0 each group was intranasally infected (I) with 1 � 108 CFU of KP35, and survival rate, body weight, anddisease severity, as indicated, were followed over 7 days. Survival rate was evaluated with a log rank test. Disease severity levels betweenIL-10�/� mice (untransferred [UT]) and IL-10�/� mice that received IL-10/GFP BM-MDSCs by transfer were compared by two-way ANOVAwith a Tukey posttest.

IL-10 from M-MDSCs in K. pneumoniae ST258 Pneumonia Infection and Immunity

May 2019 Volume 87 Issue 5 e00665-18 iai.asm.org 9

on October 12, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

FIG 9 Graphical summary of major differences of the immune responses and the role of IL-10 during KP35 andKPPR1 pneumonia. Major differences in the immune responses against sublethal doses of KPPR1 and KP35 havebeen described in WT and IL-10�/� mice. (A) The immune response against KPPR1 is characterized by a rapidbacterial clearance and a rapid infiltration of neutrophils but a much slower infiltration of M-MDSCs at 72 hpi. WTmice infected with KPPR1 produce TNF-� and IL-6 from 48 hpi to at least 96 hpi. (B) During KP35 pneumonia theimmune response is characterized by early recruitment of neutrophils and M-MDSCs able to produce IL-10 andother suppressive effectors. As a consequence, these mice present weaker production of proinflammatorycytokines and a high but decreasing bacterial burden in the lung tissue. Incredibly, these mice are very resistantto KP35 and CRKP-ST258. (C) In the absence of IL-10 the immune response against KPPR1 is more efficient,

(Continued on next page)

Peñaloza et al. Infection and Immunity

May 2019 Volume 87 Issue 5 e00665-18 iai.asm.org 10

on October 12, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

identified as the main IL-10-producing cells (23). IL-10 induction is rapid, and high levelscan be detected at 48 hpi when Ly6C� monocytes are absent from the lung tissue (21).In contrast to pulmonary infection with Mycobacterium bovis in which the early pro-duction of IL-10 is driven mostly by neutrophils at 24 hpi (24), during KPPR1 infectionrecruited MDSCs were identified as an important IL-10 source but not until 72 hpi whenthe infection was being resolved (7). The immune response to CRKP-ST258 is distinctfrom responses to these other infections; once M-MDSCs are recruited to the lung tissueat 48 hpi, they immediately start the production of IL-10 and continue for at least10 dpi. This kinetic of IL-10-producing MDSCs is consistent with the reduced productionof proinflammatory cytokines at 48 hpi observed in WT but not IL-10�/� mice. Our dataare consistent with previous reports that describe the ability of IL-10 to inhibit TNF-�and IL-12 production (25, 26) and IL-1� through the suppression of the NLRP3-inflammasome (27).

IL-10 has a major role in protecting the host from damaging inflammation. Inresponse to bacterial infection, MDSC recruitment and activity are typically associatedwith resolution and/or chronicity (28). This has been described during pulmonaryinfections caused by Pseudomonas aeruginosa (28) and nonpulmonary infections withmajor pathogens such as Staphylococcus aureus, Salmonella enterica, and Porphyromo-nas gingivalis (10, 13, 14, 29–34). In each of these cases, recruited MDSCs inhibit theinnate and adaptive immune responses, causing chronic infection. This immunosup-pressive role of IL-10-producing MDSCs in the lungs is important during lipopolysac-charide (LPS) endotoxic shock and post-influenza pneumococcal pneumonia (35). IL-10also affects other types of immune cells. IL-10 production protects B cells from death,improving their antigen presentation and antibody isotype change (36–39). The rele-vance of B cell function in K. pneumoniae infections was demonstrated in a recent studythat showed that capsule antibodies enhance the serum-mediated clearance of CRKP-ST258 and promote neutrophil-mediated phagocytic clearance of these organisms (40).

In our model, we provide strong evidence that the production of IL-10 by M-MDSCsand also by other myeloid cells is a major mechanism of lung protection duringCRKP-ST258 pneumonia. The data presented give important insights into how IL-10modulates lung injury; first, lung hematoxylin and eosin (H&E) staining and furtheranalyses led us to identify major differences in the area compromised, the luminalexudate and parenchyma pneumonia in lungs from WT and IL-10�/� mice during thefirst 48 h. Total BALF proteins, another approximation of lung injury, were found athigher concentration in BALF of IL-10�/� mice during the first 48 h. When we lookedat proinflammatory cytokine production, IL-10�/� mice presented elevated levels ofproinflammatory cytokines involved in lung permeabilization and injury such as TNF-�,IL-1�, IL-6, and IL-12p40. Finally, the fact that transfer of BM-MDSCs able to produceIL-10 reduced lung inflammation in IL-10�/� mice in response to KP35 pneumonia gaveimportant information regarding the key role of these cells and IL-10 in the modulationof lung injury.

It is quite remarkable that other cells such as Ly6C� monocytes and eosinophils arealso important sources of IL-10 during KP35 pneumonia. Ly6C� monocytes, also knownas alternative or nonclassical monocytes (41), have been identified as important cellsinvolved in wound repair (41, 42). Specifically, nonclassical monocytes can differentiateinto anti-inflammatory macrophages (42) and promote the repair of the endothelium,disrupted either by neutrophil extravasation or by excessive production of TNF-� orIL-1� (43). The increased amount of nonclassical monocytes observed in the lung tissueand the BALF of IL-10�/� mice at 48 hpi could explain why these mice show reduced

FIG 9 Legend (Continued)presenting enhanced bacterial clearance in the first 72 hpi, increased neutrophil activation and proinflammatorycytokine production, and enhanced survival compared to levels in mice able to produce IL-10. (D) On the otherhand, in the absence of IL-10, KP35 infection leads to a diminished bacterial clearance capacity in the first 48 hpiand equivalent numbers of neutrophils and M-MDSCs but impaired neutrophil activation and increased proin-flammatory cytokine production during the first 48 hpi, with around 90% mortality at 4 dpi.

IL-10 from M-MDSCs in K. pneumoniae ST258 Pneumonia Infection and Immunity

May 2019 Volume 87 Issue 5 e00665-18 iai.asm.org 11

on October 12, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

TUNEL signal and MPO activity compared to levels in WT mice at 48 hpi. Whether thesecells are modulating cell apoptosis or neutrophil activity, their suppressive activitiesappear to be IL-10 independent. More experiments need to be done to establish thereal contribution of nonclassical monocytes during CRKP-ST258 pneumonia. On theother hand, suppressive eosinophils, called Eo-MDSCs, have been recently identifiedduring S. aureus infection (10). These cells suppress T cell function through theproduction of nitric oxide (10). It is not clear whether the eosinophils identified in ourmodel can be defined as Eo-MDSCs, and more studies are needed to establish the realcontribution of these cells in the modulation of the inflammatory response duringCRKP-ST258 pneumonia.

The increased survival and bacterial clearance observed in the lung tissue ofIL-10�/� mice receiving a transfer of IL-10-producing BM-MDSCs confirm the relevantrole of these cells during KP35 pneumonia. However, the fact that the transfer of thesecells did not reduce the production of proinflammatory cytokines also indicates that thetransient IL-10 production by other cells such as neutrophils, Ly6C� monocytes, andeosinophils is required to achieve an optimal and efficient immune response in termsof host survival, lung damage, and bacterial clearance.

Finally, even though the transfer of non-IL-10-producing BM-MDSCs did not have aneffect on bacterial clearance and lung inflammation, it had a positive impact on hostsurvival by either delaying host mortality or improving host survival from 0% to 25%.It has been reported that BM-MDSCs can efficiently suppress T cell activation throughthe activity of arginase-1, iNOS, Cox2, vascular endothelial growth factor (VEGF), Ido1,and other factors (28). It is highly possible that these molecules also have an importantrole in the modulation of the inflammatory response during KP35 infection; however,more studies are needed to establish the real contribution and the mechanismsinvolved.

CRKP-ST258 infections have become a common and challenging clinical problem. Asillustrated by the accumulating data from murine models that appear to reflect what isobserved in patients, these organisms are highly resistant to normal immune clearanceand often cause persistent infections. Our data demonstrate that during KP35 pneu-monia, the production of IL-10 by the recruited MDSC population is critical in themodulation of the proinflammatory immune response and in the prevention of hostmortality although persistent infection is promoted.

MATERIALS AND METHODSMice. C57BL/6 wild-type (WT) mice and B6.129P2-Il10tm1Cgn (IL-10�/�) and B6(Cg)-Il10tm1.1Karp

(IL-10-GFP) mice were originally obtained from The Jackson Laboratories (Bar Harbor, ME) and main-tained in the animal facility of Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile.All experimental protocols were reviewed and approved by the Scientific Ethical Committee for Animaland Environment Care (Comité Etico Científico en Cuidado Animal y Ambiente) of the PontificiaUniversidad Católica de Chile (protocols number 150721004 and 150721005). All animal work wasperformed according to the Guide for Care and Use of Laboratory Animals (44). Experiments using animalswere overseen by trained personnel daily, and a clinical score was evaluated as previously described(21, 45).

Klebsiella pneumoniae infection. KP35 was grown on Luria-Bertani (LB) medium until an opticaldensity of 0.5 was reached. Then, 1 ml was centrifuged and resuspended at a final concentration of1 � 108 CFU on 50 �l of phosphate-buffered saline (PBS). Six to 8-week-old WT, IL-10�/�, and IL-10/GFPVertX mice were anesthetized with a ketamine (80 mg/kg)-xylazine (4 mg/kg) solution and intranasallyinfected. Body weight was recorded daily; animals that lost more than 20% of their original weight wereeuthanized.

BALF collection. One milliliter of bronchoalveolar lavage fluid (BALF) was obtained from uninfectedand infected mice at 24 and 48 h postinfection (hpi) by instilling 2 ml of sterile 1� PBS into a cannulatedmouse trachea. Then the cellular fraction was separated from the supernatant by centrifugation at1,800 rpm for 20 min. The cellular fraction was used to measure the infiltrating immune cells, andsupernatant was used to quantify cytokine production, MPO activity, and total protein.

Flow cytometry. Whole lungs were recovered at different times postinfection, minced with sterilescissors, and incubated in PBS-collagenase (1 mg/ml) for 1 h at 37°C, with agitation. Homogenized lungswere filtered using a 70-�m-pore-size filter, and cells were recovered by centrifugation in sterile 1� PBSand washed once with ammonium chloride-potassium (ACK) lysis buffer and once with PBS. In parallel,cells from bronchoalveolar lavage fluid (BALF) were recovered by centrifugation and washed with ACKlysis buffer. Then, cells from lungs and BALF were stained with Live/Dead fixable viability stain 510 (BD

Peñaloza et al. Infection and Immunity

May 2019 Volume 87 Issue 5 e00665-18 iai.asm.org 12

on October 12, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

Biosciences) in PBS. Cells were washed twice, resuspended in PBS–2% fetal bovine serum (FBS), andstained with the following mix of antibodies: CD45-BV786 (BD Biosciences), CD11b-phycoerythrin (PE)(BD Biosciences), Siglec-F-PE-CF594 (BD Biosciences), Ly6G-Alexa Fluor 700 (Biolegend), CD11c-PE-Cy7(BD Biosciences), MHC-II–BV650 (BD Biosciences), CD64-Alexa Fluor 647 (Biolegend), Ly6C-BV605 (BDBiosciences), and CD24-BV421 (BD Biosciences). Before analysis, CountBright absolute counting beads(Life Technologies) were added to quantify each cell population. The gating strategy used to identifyeach cell type is described in Fig. S6 in the supplemental material.

Lung histopathological analysis. Complete lungs were removed from mice at 24 and 48 hpi, fixedin 4% paraformaldehyde, dehydrated through the exposure to different concentrations of ethanol, andinfiltrated with paraffin wax. Then, lung tissue was embedded in paraffin wax blocks. Paraffin blocks weresliced with a conventional microtome in 5-�m sections with an angle blade of 10°. Then, cut slices wereplaced at 60°C for 1 h, mounted in glass slides, and stained with hematoxylin and eosin (H&E) and TUNEL.Once stained, samples were analyzed in a Leica SCN400 slide scanner for digital imaging (magnification,�40) or in an Olympus BX51 light microscope at a magnification of �10; total lung digital images werecollected and analyzed with the ImageScope, version 11, software and ImageJ software, respectively.TUNEL staining was quantified as the percentage of TUNEL-positive area in each group using ImageJsoftware (https://imagej.nih.gov/ij/docs/examples/stained-sections/index.html). Lung damage was quan-tified in H&E-stained sections by a pathologist according to parameters listed in Table S1.

Protein and cytokine quantification in BALF. BALF from IL-10�/� and WT mice was collected andcentrifuged at 1,800 rpm for 15 min. BALF supernatant was collected and stored at �80°C until used.Total BALF protein content was measured by a conventional Bradford protein assay. Levels of IL-10,TNF-�, IL-6, IL-1�, IL-12p40, and IFN-� (in picograms/milliliter) were measured on a Luminex 200instrument (Merck Millipore), using a mouse magnetic Luminex screening assay (R&D Systems), accord-ing to the manufacturer’s instructions.

MPO activity in BALF. Myeloperoxidase (MPO) chlorination activity was evaluated in BALF using anEnzchek MPO activity assay kit according to the manufacturer’s instructions. Briefly, in a 96-well plate, BALFobtained from uninfected and infected WT and IL-10�/� mice was exposed to a mixture of 5 mM H2O2 andthe probe 3=-(p-aminophenyl) fluorescein (APF) at room temperature in the dark for 10 min. Fluorescence(emission wavelength, 530 nm; excitation wavelength, 485 nm) was quantified in a Cytation3 plate reader.

Bone marrow-derived MDSC generation, adoptive transfer, and bactericidal capacity. Bonemarrow MDSCs were generated as previously described (16, 17) with some modifications. Briefly, totalbone marrow cells were purified from donor IL-10�/� and IL-10/GFP VertX mice. Cells were counted, and2.5 � 106 bone marrow cells were cultured for 5 days in 100-mm culture dishes with 10 ml of RPMI 1640medium supplemented with 2 mM L-glutamine, 10 mM HEPES, 20 �M 2-mercaptoethanol (2-ME), 1%streptomycin-penicillin, 10% heat-inactivated FBS, and 40 ng/ml of G-CSF and GM-CSF. After 5 days, cellswere washed with sterile 1� PBS and counted. A total of 1 � 106 cells were intranasally transferred toreceptor IL-10�/� and IL-10/GFP VertX mice. At 24 h posttransfer, mice were intranasally infected with1 � 108 CFU of KP35. Survival rate and severity of disease were followed over 7 days. Before the adoptivetransfer, BM-MDSCs were stained and phenotypically characterized by flow cytometry using the follow-ing antibodies: CD11b-peridinin chlorophyll protein (PerCP)-Cy5 (BD), Ly6C-BV605 (BD), Ly6G-AF700(BioLegend), F4/80-allophycocyanin (APC) (BD), CD115-PE (BD), MHC-II-BV650 (BD), and CD11c-PE-Cy7(BD). To evaluate the bactericidal capacity of BM-MDSCs, 105 BM-MDSCs were seeded in 24-well platesfor 1 h at 37°C to allow cell adherence. After 1 h, BM-MDSCs were infected with KP35 (multiplicity ofinfection [MOI] of 1), and at 4 h postinfection the cells were placed on ice and lysed with distilled H2O.Serial dilutions were seeded on LB agar, and plates were incubated overnight.

Statistical analyses. A one-way analysis of variance (ANOVA) test followed by a Tukey multiple-comparison test was performed to analyze IL-10-producing cells, total BALF protein concentration, andbacterial burden in BALF, lung, and spleen over time in IL-10/GFP-expressing infected mice and for all thecomparisons for BM-MDSC transfer experiments. Survival curves were compared using a log rank test. In allcases, a P value of �0.05 was considered statistically significant. Two-way ANOVA, followed by Tukeymultiple-comparison tests, was performed to analyze cellular infiltration, lung histopathology, cytokineproduction, and MPO activity in WT and IL-10�/� mice at 24 and 48 hpi. Bacterial burdens in lung, BALF, andspleen between WT and IL-10�/� mice were compared with a Student t test (P value of �0.05). Allcomparisons were performed using the GraphPad Prism software, version 7.0a, for Macintosh (GraphPadsoftware).

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at https://doi.org/10.1128/IAI

.00665-18.SUPPLEMENTAL FILE 1, PDF file, 2.2 MB.

ACKNOWLEDGMENTSThis study was supported by grants from Fondo Nacional de Ciencia y Tecnología de

Chile (grant no. 1170964), the Millennium Institute on Immunology and Immunother-apy (P09/016-F), the Comisión Nacional de Investigación Científica y Tecnológica (grantno. 21140214), and NIH (R35 HL135800 and NIH K08 HL138289).

We thank Luis Larrondo and Consuelo Olivares, from the Faculty of BiologicalSciences, Pontificia Universidad Católica de Chile, for providing access to the plate

IL-10 from M-MDSCs in K. pneumoniae ST258 Pneumonia Infection and Immunity

May 2019 Volume 87 Issue 5 e00665-18 iai.asm.org 13

on October 12, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

reader Cytation 3 and Alexis Kalergis from the Faculty of Biological Sciences, PontificiaUniversidad Católica de Chile, for providing access to the flow cytometer BDFortessa.

We declare that we have no financial conflicts of interest.

REFERENCES1. Nordmann P, Cuzon G, Naas T. 2009. The real threat of Klebsiella pneu-

moniae carbapenemase-producing bacteria. Lancet Infect Dis9:228 –236. https://doi.org/10.1016/S1473-3099(09)70054-4.

2. Munoz-Price LS, Poirel L, Bonomo RA, Schwaber MJ, Daikos GL, Cormican M,Cornaglia G, Garau J, Gniadkowski M, Hayden MK, Kumarasamy K, Liver-more DM, Maya JJ, Nordmann P, Patel JB, Paterson DL, Pitout J, Villegas MV,Wang H, Woodford N, Quinn JP. 2013. Clinical epidemiology of the globalexpansion of Klebsiella pneumoniae carbapenemases. Lancet Infect Dis 13:785–796. https://doi.org/10.1016/S1473-3099(13)70190-7.

3. Paczosa MK, Mecsas J. 2016. Klebsiella pneumoniae: going on the offensewith a strong defense. Microbiol Mol Biol Rev 80:629 – 661. https://doi.org/10.1128/MMBR.00078-15.

4. Ahn D, Peñaloza H, Wang Z, Wickersham M, Parker D, Patel P, KollerA, Chen EI, Bueno SM, Uhlemann AC, Prince A. 2016. Acquiredresistance to innate immune clearance promotes Klebsiella pneu-moniae ST258 pulmonary infection. JCI Insight 1:e89704. https://doi.org/10.1172/jci.insight.89704.

5. Kobayashi SD, Porter AR, Dorward DW, Brinkworth AJ, Chen L, KreiswirthBN, DeLeo FR. 2016. Phagocytosis and killing of carbapenem-resistantST258 Klebsiella pneumoniae by human neutrophils. J Infect Dis 213:1615–1622. https://doi.org/10.1093/infdis/jiw001.

6. Gabrilovich DI. 2017. Myeloid-derived suppressor cells. Cancer ImmunolRes 5:3– 8. https://doi.org/10.1158/2326-6066.CIR-16-0297.

7. Poe SL, Arora M, Oriss TB, Yarlagadda M, Isse K, Khare A, Levy DE, Lee JS,Mallampalli RK, Chan YR, Ray A, Ray P. 2013. STAT1-regulated lungMDSC-like cells produce IL-10 and efferocytose apoptotic neutrophilswith relevance in resolution of bacterial pneumonia. Mucosal Immunol6:189 –199. https://doi.org/10.1038/mi.2012.62.

8. Gabrilovich DI, Nagaraj S. 2009. Myeloid-derived suppressor cells asregulators of the immune system. Nat Rev Immunol 9:162–174. https://doi.org/10.1038/nri2506.

9. Bronte V, Brandau S, Chen SH, Colombo MP, Frey AB, Greten TF, Mandru-zzato S, Murray PJ, Ochoa A, Ostrand-Rosenberg S, Rodriguez PC, Sica A,Umansky V, Vonderheide RH, Gabrilovich DI. 2016. Recommendations formyeloid-derived suppressor cell nomenclature and characterization stan-dards. Nat Commun 7:12150. https://doi.org/10.1038/ncomms12150.

10. Goldmann O, Beineke A, Medina E. 2017. Identification of a novel subsetof myeloid-derived suppressor cells during chronic staphylococcal infec-tion that resembles immature eosinophils. J Infect Dis 216:1444 –1451.https://doi.org/10.1093/infdis/jix494.

11. Penaloza HF, Schultz BM, Nieto PA, Salazar GA, Suazo I, Gonzalez PA,Riedel CA, Alvarez-Lobos MM, Kalergis AM, Bueno SM. 2016. Opposingroles of IL-10 in acute bacterial infection. Cytokine Growth Factor Rev32:17–30. https://doi.org/10.1016/j.cytogfr.2016.07.003.

12. Cyktor JC, Turner J. 2011. Interleukin-10 and immunity against prokary-otic and eukaryotic intracellular pathogens. Infect Immun 79:2964 –2973.https://doi.org/10.1128/IAI.00047-11.

13. Tam JW, Kullas AL, Mena P, Bliska JB, van der Velden AW. 2014. CD11b�Ly6Chi Ly6G- immature myeloid cells recruited in response to Salmonellaenterica serovar Typhimurium infection exhibit protective and immuno-suppressive properties. Infect Immun 82:2606 –2614. https://doi.org/10.1128/IAI.01590-13.

14. Tebartz C, Horst SA, Sparwasser T, Huehn J, Beineke A, Peters G, MedinaE. 2015. A major role for myeloid-derived suppressor cells and a minorrole for regulatory T cells in immunosuppression during Staphylococcusaureus infection. J Immunol 194:1100 –1111. https://doi.org/10.4049/jimmunol.1400196.

15. Gomez-Simmonds A, Greenman M, Sullivan SB, Tanner JP, Sowash MG,Whittier S, Uhlemann AC. 2015. Population structure of Klebsiella pneu-moniae causing bloodstream infections at a New York City tertiary carehospital: diversification of multidrug-resistant isolates. J Clin Microbiol53:2060 –2067. https://doi.org/10.1128/JCM.03455-14.

16. Highfill SL, Rodriguez PC, Zhou Q, Goetz CA, Koehn BH, Veenstra R,Taylor PA, Panoskaltsis-Mortari A, Serody JS, Munn DH, Tolar J, OchoaAC, Blazar BR. 2010. Bone marrow myeloid-derived suppressor cells(MDSCs) inhibit graft-versus-host disease (GVHD) via an arginase-1-

dependent mechanism that is up-regulated by interleukin-13. Blood116:5738 –5747. https://doi.org/10.1182/blood-2010-06-287839.

17. Marigo I, Bosio E, Solito S, Mesa C, Fernandez A, Dolcetti L, Ugel S, SondaN, Bicciato S, Falisi E, Calabrese F, Basso G, Zanovello P, Cozzi E, Man-druzzato S, Bronte V. 2010. Tumor-induced tolerance and immune sup-pression depend on the C/EBP� transcription factor. Immunity 32:790 – 802. https://doi.org/10.1016/j.immuni.2010.05.010.

18. Tzouvelekis LS, Miriagou V, Kotsakis SD, Spyridopoulou K, Athanasiou E,Karagouni E, Tzelepi E, Daikos GL. 2013. KPC-producing, multidrug-resistantKlebsiella pneumoniae sequence type 258 as a typical opportunistic patho-gen. Antimicrob Agents Chemother 57:5144–5146. https://doi.org/10.1128/AAC.01052-13.

19. Xiong H, Carter RA, Leiner IM, Tang YW, Chen L, Kreiswirth BN, Pamer EG.2015. Distinct contributions of neutrophils and CCR2� monocytes topulmonary clearance of different Klebsiella pneumoniae strains. InfectImmun 83:3418 –3427. https://doi.org/10.1128/IAI.00678-15.

20. Lawlor MS, Hsu J, Rick PD, Miller VL. 2005. Identification of Klebsiellapneumoniae virulence determinants using an intranasal infection model.Mol Microbiol 58:1054 –1073. https://doi.org/10.1111/j.1365-2958.2005.04918.x.

21. Peñaloza HF, Nieto PA, Muñoz-Durango N, Salazar-Echegarai FJ, Torres J,Parga MJ, Alvarez-Lobos M, Riedel CA, Kalergis AM, Bueno SM. 2015.Interleukin-10 plays a key role in the modulation of neutrophils recruit-ment and lung inflammation during infection by Streptococcus pneu-moniae. Immunology 146:100 –112. https://doi.org/10.1111/imm.12486.

22. Greenberger MJ, Strieter RM, Kunkel SL, Danforth JM, Goodman RE,Standiford TJ. 1995. Neutralization of IL-10 increases survival in a murinemodel of Klebsiella pneumonia. J Immunol 155:722–729.

23. Duell BL, Tan CK, Carey AJ, Wu F, Cripps AW, Ulett GC. 2012. Recentinsights into microbial triggers of interleukin-10 production in the hostand the impact on infectious disease pathogenesis. FEMS Immunol MedMicrobiol 64:295–313. https://doi.org/10.1111/j.1574-695X.2012.00931.x.

24. Chung EY, Liu J, Homma Y, Zhang Y, Brendolan A, Saggese M, Han J,Silverstein R, Selleri L, Ma X. 2007. Interleukin-10 expression in macro-phages during phagocytosis of apoptotic cells is mediated by home-odomain proteins Pbx1 and Prep-1. Immunity 27:952–964. https://doi.org/10.1016/j.immuni.2007.11.014.

25. Armstrong L, Jordan N, Millar A. 1996. Interleukin 10 (IL-10) regulation oftumour necrosis factor alpha (TNF-alpha) from human alveolar macro-phages and peripheral blood monocytes. Thorax 51:143–149. https://doi.org/10.1136/thx.51.2.143.

26. Rahim SS, Khan N, Boddupalli CS, Hasnain SE, Mukhopadhyay S. 2005.Interleukin-10 (IL-10) mediated suppression of IL-12 production in RAW264.7 cells also involves c-rel transcription factor. Immunology 114:313–321. https://doi.org/10.1111/j.1365-2567.2005.02107.x.

27. Yao Y, Vent-Schmidt J, McGeough MD, Wong M, Hoffman HM, Steiner TS,Levings MK. 2015. Tr1 cells, but not Foxp3� regulatory t cells, suppressNLRP3 inflammasome activation via an IL-10-dependent mechanism. JImmunol 195:488–497. https://doi.org/10.4049/jimmunol.1403225.

28. Peñaloza HF, Álvarez D, Muñoz-Durango N, Schultz BM, González PA,Kalergis AM, Bueno SM. 2018. The role of myeloid-derived suppressorcells in chronic infectious diseases and the current methodology avail-able for their study. J Leukoc Biol https://doi.org/10.1002/JLB.MR0618-233R.

29. Rieber N, Brand A, Hector A, Graepler-Mainka U, Ost M, Schafer I, WeckerI, Neri D, Wirth A, Mays L, Zundel S, Fuchs J, Handgretinger R, Stern M,Hogardt M, Doring G, Riethmuller J, Kormann M, Hartl D. 2013. Flagellininduces myeloid-derived suppressor cells: implications for Pseudomonasaeruginosa infection in cystic fibrosis lung disease. J Immunol 190:1276 –1284. https://doi.org/10.4049/jimmunol.1202144.

30. Heim CE, Vidlak D, Scherr TD, Kozel JA, Holzapfel M, Muirhead DE, KielianT. 2014. Myeloid-derived suppressor cells contribute to Staphylococcusaureus orthopedic biofilm infection. J Immunol 192:3778 –3792. https://doi.org/10.4049/jimmunol.1303408.

31. Heim CE, Vidlak D, Kielian T. 2015. Interleukin-10 production by myeloid-derived suppressor cells contributes to bacterial persistence during

Peñaloza et al. Infection and Immunity

May 2019 Volume 87 Issue 5 e00665-18 iai.asm.org 14

on October 12, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

Staphylococcus aureus orthopedic biofilm infection. J Leukoc Biol 98:1003–1013. https://doi.org/10.1189/jlb.4VMA0315-125RR.

32. Oz HH, Zhou B, Voss P, Carevic M, Schroth C, Frey N, Rieber N, Hector A,Hartl D. 2016. Pseudomonas aeruginosa airway infection recruits andmodulates neutrophilic myeloid-derived suppressor cells. Front Cell In-fect Microbiol 6:167. https://doi.org/10.3389/fcimb.2016.00167.

33. Su L, Xu Q, Zhang P, Michalek SM, Katz J. 2017. Phenotype and functionof myeloid-derived suppressor cells induced by Porphyromonas gingiva-lis infection. Infect Immun 85:e00213-17. https://doi.org/10.1128/IAI.00213-17.

34. Yamada KJ, Heim CE, Aldrich AL, Gries CM, Staudacher AG, Kielian T.2018. Arginase-1 expression in myeloid cells regulates S. aureus plank-tonic but not biofilm infection. Infect Immun 86:e00206-18. https://doi.org/10.1128/IAI.00206-18.

35. Namkoong H, Ishii M, Fujii H, Yagi K, Asami T, Asakura T, Suzuki S, HegabAE, Kamata H, Tasaka S, Atarashi K, Nakamoto N, Iwata S, Honda K, KanaiT, Hasegawa N, Koyasu S, Betsuyaku T. 2018. Clarithromycin expandsCD11b� Gr-1� cells via the STAT3/Bv8 axis to ameliorate lethal endo-toxic shock and post-influenza bacterial pneumonia. PLoS Pathog 14:e1006955. https://doi.org/10.1371/journal.ppat.1006955.

36. Malisan F, Briere F, Bridon JM, Harindranath N, Mills FC, Max EE, BanchereauJ, Martinez-Valdez H. 1996. Interleukin-10 induces immunoglobulin G iso-type switch recombination in human CD40-activated naive B lymphocytes.J Exp Med 183:937–947. https://doi.org/10.1084/jem.183.3.937.

37. Shparago N, Zelazowski P, Jin L, McIntyre TM, Stuber E, Pecanha LM,Kehry MR, Mond JJ, Max EE, Snapper CM. 1996. IL-10 selectively regu-lates murine Ig isotype switching. Int Immunol 8:781–790. https://doi.org/10.1093/intimm/8.5.781.

38. Gary-Gouy H, Harriague J, Bismuth G, Platzer C, Schmitt C, Dalloul AH.

2002. Human CD5 promotes B-cell survival through stimulation of au-tocrine IL-10 production. Blood 100:4537– 4543. https://doi.org/10.1182/blood-2002-05-1525.

39. Tangye SG, Ferguson A, Avery DT, Ma CS, Hodgkin PD. 2002. Isotypeswitching by human B cells is division-associated and regulated bycytokines. J Immunol 169:4298 – 4306. https://doi.org/10.4049/jimmunol.169.8.4298.

40. Kobayashi SD, Porter AR, Freedman B, Pandey R, Chen L, Kreiswirth BN,DeLeo FR. 2018. Antibody-mediated killing of carbapenem-resistantST258 Klebsiella pneumoniae by human neutrophils. mBio 9:e00297-18.https://doi.org/10.1128/mBio.00297-18.

41. Dal-Secco D, Wang J, Zeng Z, Kolaczkowska E, Wong CH, Petri B,Ransohoff RM, Charo IF, Jenne CN, Kubes P. 2015. A dynamic spectrumof monocytes arising from the in situ reprogramming of CCR2� mono-cytes at a site of sterile injury. J Exp Med 212:447– 456. https://doi.org/10.1084/jem.20141539.

42. Olingy CE, San Emeterio CL, Ogle ME, Krieger JR, Bruce AC, Pfau DD,Jordan BT, Peirce SM, Botchwey EA. 2017. Non-classical monocytes arebiased progenitors of wound healing macrophages during soft tissueinjury. Sci Rep 7:447. https://doi.org/10.1038/s41598-017-00477-1.

43. Ginhoux F, Jung S. 2014. Monocytes and macrophages: developmentalpathways and tissue homeostasis. Nat Rev Immunol 14:392– 404. https://doi.org/10.1038/nri3671.

44. National Research Council. 1996. Guide for the care and use of labora-tory animals. National Academy Press, Washington, DC.

45. Mook-Kanamori B, Geldhoff M, Troost D, van der Poll T, van de Beek D.2012. Characterization of a pneumococcal meningitis mouse model.BMC Infect Dis 12:71. https://doi.org/10.1186/1471-2334-12-71.

IL-10 from M-MDSCs in K. pneumoniae ST258 Pneumonia Infection and Immunity

May 2019 Volume 87 Issue 5 e00665-18 iai.asm.org 15

on October 12, 2020 by guest

http://iai.asm.org/

Dow

nloaded from