Distinct Contributions of Neutrophils and CCR2⁺ Monocytes to Pulmonary Clearance of Different Klebsiella pneumoniae Strains

Abstract

Klebsiella pneumoniae is a common respiratory pathogen, with some strains having developed broad resistance to clinically available antibiotics. Humans can become infected with many different K. pneumoniae strains that vary in genetic background, antibiotic susceptibility, capsule composition, and mucoid phenotype. Genome comparisons have revealed differences between K. pneumoniae strains, but the impact of genomic variability on immune-mediated clearance of pneumonia remains unclear. Experimental studies of pneumonia in mice have used the rodent-adapted 43816 strain of K. pneumoniae and demonstrated that neutrophils are essential for optimal host defense. It remains unclear, however, whether CCR2⁺ monocytes contribute to K. pneumoniae clearance from the lung. We selectively depleted neutrophils, CCR2⁺ monocytes, or both from immunocompetent mice and determined susceptibility to infection by the 43816 strain and 4 newly isolated clinical K. pneumoniae strains. The clinical K. pneumoniae strains, including one carbapenem-resistant ST258 strain, are less virulent than 43816. Optimal clearance of each of the 5 strains required either neutrophils or CCR2⁺ monocytes. Selective neutrophil depletion markedly worsened infection with K. pneumoniae strain 43816 and three clinical isolates but did not increase susceptibility of mice to infection with the carbapenem-resistant K. pneumoniae ST258 strain. Depletion of CCR2⁺ monocytes delayed recovery from infection with each of the 5 K. pneumoniae strains, revealing a contribution of these cells to bacterial clearance from the lung. Our findings demonstrate strain-dependent variation in the contributions of neutrophils and CCR2⁺ monocytes to clearance of K. pneumoniae pulmonary infection.

INTRODUCTION

Klebsiella pneumoniae is a common cause of pneumonia, particularly in elderly and immunocompromised patients requiring prolonged hospitalization (1). Over the past 20 years, antibiotic resistance among K. pneumoniae strains has become increasingly problematic, reaching a new plateau in the past decade with the discovery and subsequent international dissemination of strains with high-level carbapenem resistance (2, 3). Treatment of highly antibiotic-resistant K. pneumoniae infections is limited by the paucity and toxicity of effective antibiotics, and failure to cure is common and accounts for the high mortality associated with systemic infection by this pathogen. In patients, susceptibility to K. pneumoniae infections is associated with defective pulmonary clearance mechanisms, as occur during endotracheal intubation, and deficient neutrophil function (1).

K. pneumoniae lung infection has been studied extensively in a mouse model using the rodent-adapted 43816 strain. Studies with a wide range of gene-knockout mice demonstrate that lung macrophages and neutrophils contribute to resolution of K. pneumoniae infection (4, 5); however, the relative contributions that these cells make remain unclear. Studies of chemokine receptor-deficient mice demonstrated that reduced recruitment of neutrophils increases susceptibility to K. pneumoniae 43816 infection (6–8). However, in many of these studies, inflammatory monocytes (IMs) and neutrophils could not be distinguished because sufficiently sensitive reagents were not available. Along similar lines, mice lacking MyD88, TRIF, interleukin-1 receptor (IL-1R), Toll-like receptor 4 (TLR4), and leukotriene B₄ (LTB4) had reduced neutrophil recruitment and reduced resistance to K. pneumoniae 43816 infection, but the impact of these deficiencies on inflammatory monocyte recruitment remains unclear (6, 9–11). In other cases, such as deficiency of the C-type lectin receptors Clec4E and Clec4D, resistance to K. pneumoniae infection was reduced but neutrophil recruitment to the lung was increased (12, 13). The demonstration that IL-17R deficiency increases susceptibility to K. pneumoniae infection further supports the potential role of neutrophils in bacterial clearance (14).

K. pneumoniae is an encapsulated, Gram-negative rod that is predominantly extracellular during lung infection. Its principal residence in the mammalian host is the lower gastrointestinal tract, where it generally constitutes a minor population that can undergo marked expansion when commensal microbiota diversity is reduced by antibiotic treatment (15). A variety of systemic infections are caused by K. pneumoniae, including hepatic abscess, cholangitis, and bacterial sepsis and pneumonia (16–18). K. pneumoniae virulence is largely dependent on the expression of the polysaccharide capsule, and different K. pneumoniae strains express distinct capsule types (19). The association between a specific capsule type and hepatic abscess development suggests that capsule composition may contribute to and potentially determine the pathogenesis of K. pneumoniae infection (18). The extent to which capsule types contribute to pulmonary infection and bacterial susceptibility to clearance by different inflammatory cell populations in the lung is unclear.

Inflammatory monocytes are circulating blood cells with surface expression of Ly6C and chemokine receptor CCR2. These surface markers, together with the bean-shaped, unilobular nucleus, distinguish monocytes from neutrophils, which express Ly6G and not Ly6C or CCR2. Monocytes rely on CCR2-CCL2 signaling to egress from the bone marrow (20). Although they represent a small fraction of circulating leukocytes, they are highly responsive to microbial infection and emigrate from the bone marrow to infiltrate sites of infection. There they can become activated to produce tumor necrosis factor (TNF) and inducible nitric oxide synthase (iNOS) and contribute to microbicidal responses (21). Recently, a potential role for CCR2⁺ monocytes in defense against Klebsiella pneumoniae was suggested by infection of CCR2^−/− mice with K. pneumoniae strain 43816 (22).

Here, we have used the murine model of K. pneumoniae infection to investigate the relative contributions of neutrophils and CCR2⁺ monocytes to pulmonary clearance and survival. In order to determine whether strains of K. pneumoniae differ in terms of their engagement of neutrophils and CCR2⁺ monocytes during the course of infection, we obtained 4 distinct clinical strains of K. pneumoniae isolated from the bloodstreams of patients undergoing cancer treatment at Memorial Hospital and compared them to the commonly studied, rodent-adapted 43816 strain. We found that neutrophils contributed to pulmonary clearance of 4 out of 5 strains of K. pneumoniae, while CCR2⁺ monocyte depletion worsened infection by all 5 strains. Unexpectedly, our study reveals a K. pneumoniae strain-dependent variation in the contributions of neutrophils and CCR2⁺ monocytes to bacterial clearance from the infected lung.

MATERIALS AND METHODS

Mouse experiments.

C57BL/6 (wild-type [WT] B6) mice were purchased from the Jackson Laboratory. Generation of CCR2-DTR mice was previously described (23). All mice were bred and maintained under specific-pathogen-free conditions at the Memorial Sloan Kettering Research Animal Resource Center. Sex-, age-, and weight-matched controls were used in all experiments according to institutional guidelines for animal care. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Memorial Sloan-Kettering Cancer Center. According to the animal protocol, mice that were moribund, severely ill, or had lost more than 20% of body weight were euthanized. For antibody-mediated depletion, injection by the intraperitoneal (i.p.) or intravenous (i.v.) route failed to effectively deplete target cells in infected lungs (data not shown), so we administered depletion antibody by the i.p./i.v. and intratracheal (i.t.) routes. Anti-Gr1 antibody (clone RB6-8C5) was administered i.p. (400 μg), i.v. (100 μg), and i.t. (100 μg). Anti-Ly6G antibody (clone 1A8) was given i.p. (600) μg, i.v. (200 μg), and i.t. (200 μg). Both antibodies were given on a daily basis, starting on 1 day prior to infection until the end of the experiments. For diphtheria toxin (DT)-mediated depletion of IMs, CCR2-DTR animals were injected i.p. with 25 ng/g body weight of DT every 2 days, beginning 1 day before infection.

Capsule staining.

K. pneumoniae strains were grown in milk-LB broth overnight and were plated on microscopy glass slides and briefly heat fixed. The slides were subjected to crystal violet staining. Images were taken under a magnification of ×100.

Bacterial growth conditions and quantification of bacterial burden.

Clinical isolates were obtained from the Clinical Microbiology Laboratory and derived from blood cultures from patients undergoing treatment at Memorial Hospital, Memorial Sloan Kettering Cancer Center. Strain 43816 was purchased from ATCC. All the bacteria were grown in LB medium with shaking at 37°C. Bacterial burden was quantified by plating serial dilutions of homogenized organs on LB plates. Antimicrobial susceptibility testing was with a Vitek 2 Automated Testing System by the Clinical Microbiology Service in Memorial Hospital and was interpreted according to CLSI guidelines (24).

MLST and wzi sequencing.

Multilocus sequence typing (MLST) for strain 43816 and other five clinical K. pneumoniae strains were conducted using the method described previously (25). The capsular type was detected by wzi gene sequencing using a method described elsewhere (26). wzi is a conserved gene in all capsular types of K. pneumoniae, and the sequences of wzi can be used for the prediction of K type in K. pneumoniae (26).

Intratracheal inoculation of mice.

Bacteria were grown in LB medium to log phase. Cultures were washed and diluted with phosphate-buffered saline (PBS). Animals were anesthetized by inhaling isoflurane. A blunt curved needle was inserted into the trachea through the mouth, and 50 μl of bacterial suspension was applied through the syringe and the needle. Mice were held vertically for 30 s after the inoculation.

Tissue preparation for flow cytometry and cell staining.

The whole lung was perfused before harvest, homogenized, and digested with 5% fetal calf serum (FCS), 5,000 U/ml collagenase type IV (Worthington), and 20 U/ml DNase I (Roche) at 37°C for 40 min to obtain single-cell suspensions for the purpose of staining or plating. Single-cell suspensions were stained and analyzed on a BD LSR II cytometer. Antibodies were purchased from BD Bioscience unless otherwise indicated. The following clones were used: anti-Ly6C (AL-21), Ly6G (1A8), CD11b (M1/70), CD45 (30F-11), and TNF-α (MP6-XT22). Intracellular TNF-α staining was carried out using a BD Cytofix/Cytoperm kit.

Histology.

Mice were sacrificed, and an incision was made at the trachea. The lung was inflated through the incision using 4% paraformaldehyde (PFA). The lung was then harvested and placed in 4% PFA to fix overnight at 4 degrees. The tissue was then washed with 70% ethanol and subjected to paraffin embedding and sectioning. The thickness of each section was 5 μm. The sections were stained with hematoxylin and eosin (H&E) by the Cytology Core at Memorial Sloan Kettering Cancer Center (MSKCC).

Statistical analysis and LD₅₀ determinations.

GraphPad Prism was used for statistical analysis. Bacterial burdens were compared after log transformation. Differences between means were evaluated by two-tailed unpaired t test or one-way analysis of variance (ANOVA). The following symbols were used to indicate statistical significance: n.s., nonsigfinicant (P > 0.05); *, P ≤ 0.05; **, P ≤ 0.01; and ***, P ≤ 0.001. To determine 50% lethal dose (LD₅₀) values following pulmonary infection with the 5 K. pneumoniae strains, we used the equation y (lethality) = m · x (dose) + c by determining the value of x after applying the value of 50 for y.

RESULTS

Clinical isolates of K. pneumoniae are diverse and are distinct from the 43816 laboratory strain.

In order to determine whether immune defenses against recently isolated clinical K. pneumoniae isolates and the commonly investigated 43816 laboratory strain differ, we obtained four isolates from the Memorial Hospital Clinical Microbiology Laboratory that had been recovered from blood cultures of patients receiving cancer treatment. Multilocus sequence typing (MLST) was performed on all 5 K. pneumoniae strains and revealed that each strain has a distinct sequence type (ST) (Table 1) (25). Based on the site of isolation (Memorial Hospital) and the assigned MLST number of each strain, we named the clinical isolates Kp-MH189, Kp-MH1867, Kp-MH225, and Kp-MH258. Notably, Kp-MH258 is an example of the epidemic ST258 clone, which has successfully spread globally, harboring the bla_KPC-encoded carbapenemase (27). These four strains and the laboratory strain 43816 were morphologically indistinguishable by Gram staining, and each expressed a microscopically detectable capsule (see Fig. S1A in the supplemental material). Each strain, however, had a distinct capsule type, based on wzi sequencing and its predicted K type (Table 1). Each strain was also distinct in terms of antibiotic resistance upon testing against 18 clinically relevant antibiotics (Table 2). Of note, laboratory strain 43816 was susceptible to all the tested antibiotics, while Kp-MH258 was resistant to nearly all antibiotics, including carbapenems (ertapenem and meropenem). Kp-MH189, Kp-MH1867, and Kp-MH225, in contrast, were susceptible to carbapenems but variably resistant to different β-lactam antibiotics. On the basis of MLST, capsular types, and antibiotic resistance, the 5 strains are genetically distinct.

TABLE 1.

Genotyping and capsule serotyping of the isolates^a

Type	Result for isolate:
	1	2	3	4	43816
Genotype
gapA	3	2	10	3	2
infB	2	3	1	3	1
mdh	41	2	1	1	70
pgi	1	35	1	1	1
phoE	17	1	12	1	12
rpoB	4	4	1	1	1
tonB	46	4	38	79	127
MLST	189	170	225	258	439
wzi sequence type	wzi99	wzi82	wzi59	wzi154	wzi2
K type	K31	K23-like	K3	ST258 cps-2	K2

^aThe numbers under “Genotype” are uniquely assigned to different alleles of each gene. Multilocus sequence typing (MLST) was performed with the listed housekeeping genes. wzi sequencing was used to determine the K types.

TABLE 2.

Antibiotic sensitivities of the isolates^a

^aEach bacterial strain was tested against 18 clinically relevant antibiotics. Drugs belonging to the carbapenem class are highlighted in blue. S, sensitive; R, resistant; I, intermediate.

The clinical strains are less pathogenic than the laboratory strain.

To estimate the relative virulence of each strain in the murine pneumonia model, we infected C57BL/6 mice intratracheally with each strain over a range of inoculum sizes and determined the 50% lethal dose (LD₅₀) and density of live bacteria per lung at different times following infection. The four clinical strains had LD₅₀s of between 5.6 × 10⁶ and 1.6 × 10⁷, whereas strain 43816, in concordance with previous studies (4, 7, 28), had a markedly lower LD₅₀ of 1.8 × 10⁴ (Fig. 1). Next, we measured in vivo growth and survival of the K. pneumoniae strains by inoculating C57BL/6 mice with 0.1 LD₅₀ of each strain and measuring bacterial CFU in lungs at different time points after challenge. Although all five strains grew at similar rates during in vitro culture (see Fig. S1B in the supplemental material), they differed in growth and survival in vivo: the four clinical strains reached 10⁵ to 10⁶ CFU at 24 h, whereas strain 43816 reached 10⁶ to 10⁷ CFU despite a lower initial inoculum. At 48 h, the CFU of the four clinical isolates were markedly reduced, while strain 43816 persisted in the lung in excess of 96 h but was cleared by 6 days (Fig. 2). These results demonstrate that strain 43816, in comparison to the 4 clinical isolates, has an enhanced ability to proliferate and persist in the murine lung but that all 5 strains are eventually cleared.

FIG 1.

LD₅₀s of different K. pneumoniae strains upon intratracheal inoculation of mice. WT C57BL/6 mice were inoculated intratracheally (i.t.) with increasing doses of each isolate. The survival rates were determined on day 7 postinfection, and the LD₅₀s were calculated.

FIG 2.

Time course of bacterial clearance in the lung. WT animals were inoculated i.t. with different strains at a dose of 0.1 LD₅₀. At the designated time points, the mice were sacrificed. The lungs were perfused, homogenized, and plated following serial dilution to determine the bacterial burden, which was plotted as CFU.

CCR2⁺ monocytes and neutrophils are recruited to the infected lung.

Neutrophils and CCR2⁺ monocytes are two predominant innate immune cell populations that are rapidly and often concurrently recruited to sites of infection. Both cell populations have been implicated in early defense against a wide range of bacterial pathogens (29). Intratracheal inoculation of C57BL/6 mice with each of the five K. pneumoniae strains induced rapid and robust recruitment of monocytes and neutrophils to the lungs at 24 h following infection (Fig. 3A to C). Together, these two cell populations constituted a major fraction of the total recruited immune cells, accounting for around or over 50% of CD45⁺ cells in infected lungs. Pulmonary infiltration with monocytes and neutrophils was maintained for over 48 h, albeit to a lesser degree for mice infected with the clinical strains, where bacterial CFU were reduced (Fig. 2). H&E staining of lungs demonstrated interstitial infiltration with inflammatory cells that was similar in mice infected with each of the 5 K. pneumoniae strains (Fig. 4). This interstitial pattern can be seen during early stages of infection (24 h) that precede the development of bronchopneumonia or lobar pneumonia (30, 31). Previous studies demonstrated the importance of TNF-α in defense against pneumonia and bacteremia following infection by the 43816 strain (32, 33), although the source of TNF-α remained unclear. We found that CCR2⁺ monocytes were robust producers of TNF-α in response to infection with all 5 K. pneumoniae strains (Fig. 3A). Neutrophils, on the other hand, did not generate significant levels of TNF-α (Fig. 3B). Our findings suggested that TNF-α production by recruited monocytes may contribute to the initiation of defense mechanisms against K. pneumoniae.

FIG 3.

Neutrophil and CCR2⁺ monocyte recruitment to the lung. (A) Representative flow cytometry plots showing Ly6C⁺ CD11b⁺ monocytes and histograms of TNF-α expression by activated monocytes in the lung at 24 h following infection. WT animals were inoculated i.t. with different K. pneumoniae strains at a dose of 0.1 LD₅₀. The lung was homogenized, and the cellular infiltrates were stained and analyzed by flow cytometry. Uninf, uninfected. (B) Representative flow cytometry plots showing Ly6G⁺ CD11b⁺ neutrophils and their TNF-α expression from the same experiments. (C) Quantification of neutrophil and monocyte numbers in the lung at 24 and 48 h following infection (n = 4 per group).

FIG 4.

H&E staining of infected lungs at 24 h following infection. Magnification, ×63. Scale bar, 50 μm. Blue arrows, type I pneumocytes; black arrows, type II pneumocytes; green arrow, alveolar macrophage; orange arrows, infiltrating leukocytes.

CCR2⁺ monocytes play critical roles in defense against K. pneumoniae.

Neutrophils play an important role in defense against murine pulmonary infection with strain 43816 (14). On the other hand, the role of CCR2⁺ inflammatory monocytes is less clear. In order to investigate the roles of monocytes and neutrophils in defense against the clinical K. pneumoniae strains, we used antibody- and DT receptor (DTR)-mediated cell depletion to selectively deplete granulocytes and/or CCR2⁺ monocytes (34). Specifically, we used anti-Gr1 (clone RB6-8C5), which recognizes Ly6G and Ly6C and thus depletes both neutrophils and CCR2⁺ monocytes, anti-Ly6G (clone 1A8), which selectively depletes neutrophils but leaves inflammatory monocytes intact, and DT administration to CCR2-DTR mice, which selectively depletes CCR2-expressing cells, principally inflammatory monocytes and also a subpopulation of NK cells, but leaves neutrophil populations intact (23). Anti-Gr1 and anti-Ly6G were administered i.p. and i.t. on a daily basis (see Fig. 2A in the supplemental material). Anti-Ly6G antibody reduced neutrophils by 93% and had no detectable effect on the number of Ly6C^hi monocytes. In contrast, anti-Gr1 antibody depleted about 97% of neutrophils and 80% of inflammatory monocytes (see Fig. 2B and C in the supplemental material). CCR2-DTR mice received DT every 2 days (see Fig. S2A in the supplemental material), resulting in essentially complete ablation of Ly6C^hi CCR2⁺ monocytes, while the neutrophil population remained intact (see Fig. S2B and C in the supplemental material).

To determine the contribution of neutrophils and CCR2⁺ inflammatory monocytes to early defense against pulmonary infection, mice were depleted of either neutrophils (anti-Ly6G), inflammatory monocytes [CCR2-DTR(+DT)], or both (anti-Gr1), and then intratracheally infected with each K. pneumoniae strain at a dose of 0.1 LD₅₀. Bacterial CFU were determined at 24 h postinfection. In mice infected with Kp-MH189, Kp-MH1867, and strain 43816, selective depletion of neutrophils or monocytes resulted in markedly reduced clearance of CFU from the lungs in comparison to that in undepleted control mice, and depletion of both neutrophil and monocyte populations following anti-Gr1 administration resulted in even more CFU in the lungs (Fig. 5A, B, and E). In contrast, early clearance of Kp-MH225 was reduced upon neutrophil depletion but unaffected by selective depletion of CCR2⁺ cells (Fig. 5C). Early clearance of the carbapenem-resistant Kp-MH258, on the other hand, was unaffected by selective neutrophil depletion but highly dependent on CCR2⁺ cells, as CCR2-DTR(+DT) mice and anti-Gr1-treated mice had increased CFU, while anti-Ly6G treatment did not impair bacterial clearance from the lungs (Fig. 5D). These results suggest that pulmonary clearance of K. pneumoniae infections during the first 24 h can be mediated by neutrophils or CCR2⁺ inflammatory monocytes and that infections caused by distinct K. pneumoniae strains vary in terms of the relative contributions of these two rapidly recruited innate immune cell populations.

FIG 5.

Effects of cell depletion on bacterial clearance. Animals undergoing cell depletion were inoculated i.t. with Kp-MH189 (A), Kp-MH1867 (B), Kp-MH225 (C), Kp-MH258 (D), or 43816 (E) at a dose of 0.1 LD₅₀. At 24 h following infection, lung homogenates were plated, and the relative changes in CFU were determined by comparison to the isotype control (n = 5 for each group). Statistical analysis was carried out by comparing the absolute values of CFU of each treatment to those of the isotype control.

To investigate the contributions of neutrophils and monocytes to survival and recovery from K. pneumoniae pulmonary infection at later stages of the infection, we administered anti-Gr1, anti-Ly6G, or DT for 7 days following infection, measured weight loss, and determined survival (Fig. 6A). When challenged with Kp-MH189, neutrophil-, monocyte-, or double-depleted mice all underwent greater weight loss (Fig. 6B) and had higher mortality (Fig. 7A) than control mice, indicating that both neutrophils and monocytes contributed to recovery from infection, consistent with our findings at the early, 24-h time point (Fig. 5A). Similar results were obtained when mice were infected with Kp-MH1867 and strain 43816 (Fig. 6C and F and 7B and E). Infection with strain 43816 resulted in marked weight loss and high mortality in all the three groups of monocyte/neutrophil-depleted mice. Infection of anti-Ly6G- or anti-Gr1-treated mice with Kp-MH225 also resulted in severe weight loss and high mortality (Fig. 6D and 7C), while CCR2-DTR(+DT) animals had more delayed weight loss and a mortality of 20% (Fig. 6D and 7C). Thus, while depletion of CCR2⁺ monocytes did not adversely affect infection with Kp-MH225 during the first 24 h (Fig. 5C), CCR2⁺ monocytes appear to contribute to clearance at later stages of recovery from infection. Weight loss and mortality of mice infected with Kp-MH258 were increased in anti-Gr1-treated C57BL/6 mice and DT-treated CCR2-DTR mice, whereas mice depleted of only neutrophils were similar to control mice (Fig. 6E and 7D). Thus, early clearance (Fig. 5D) and longer-term recovery from Kp-MH258 are mediated by CCR2⁺ monocytes and are independent of neutrophils. Taken together, the results indicate that neutrophils contribute to recovery from Kp-MH189, Kp-MH1867, 43816, and Kp-MH225, while CCR2⁺ monocytes contribute to recovery from all five K. pneumoniae strains.

FIG 6.

Weight changes following infection and cell depletion. (A) Schematic of the experiment. (B to F) The animals undergoing cell depletion were challenged with Kp-MH189 (B), Kp-MH1867 (C), Kp-MH225 (D), Kp-MH258 (E), or 43816 (F) at a dose of 0.1 LD₅₀. The relative weight changes of mice were determined by comparison to the initial weight on day 0 (100). Each symbol represents one treatment group. Each death, the day it occurred, and its group are indicated as symbols at the top of each graph (n = 5 for each group).

FIG 7.

Survival rates following infection and cell depletion. Mice undergoing cell depletion were inoculated with Kp-MH189 (A), Kp-MH1867 (B), Kp-MH225 (C), Kp-MH258 (D), or 43816 (E) at a dose of 0.1 LD₅₀. The survival rates on day 7 following infection were plotted (n = 5 for each group).

On day 7, we sacrificed the surviving animals and measured the CFU in their lungs. Mice in the neutrophil/monocyte depletion groups continued to harbor viable bacteria, albeit reduced in numbers in comparison to the 24-h time point (Fig. 2), while control mice had cleared infections (Fig. 8).

FIG 8.

Bacterial burden in the surviving animals from the experiment for Fig. 6 were euthanized on day 7, lung homogenates were plated, and CFU were quantified. N.A, the whole group of mice died before day 7 so it was not possible to assess the CFU at the endpoint.

DISCUSSION

Murine models of infection have been enormously helpful in identifying and characterizing virulence factors of microbial pathogens and in defining the roles of cytokines, chemokines, and a wide range of cell populations in antimicrobial immune defense. Most experimental studies of in vivo infection use specific bacterial strains to infect mice, which greatly facilitates comparisons with results from other laboratories working with the same pathogen. An important limitation of strain-specific murine studies is that human infections can be caused by many different strains of the same bacterial species, and it is unclear whether the experimental results obtained with one strain will apply to the entire range of strains. The extent to which strains of the same bacterial species can differ is becoming increasingly apparent as the number of whole-genome sequences continues to grow. Klebsiella pneumoniae strains vary in terms of genome size, capsule composition, and the expression of antibiotic resistance (27); however, it remains unclear whether immune defenses against different K. pneumoniae strains also differ. Our study demonstrates that, at least with respect to the relative contributions of neutrophils and CCR2⁺ monocytes, in vivo clearance mechanisms differ for distinct K. pneumoniae strains. Importantly, our study also demonstrates that CCR2⁺ monocyte recruitment to the lungs of infected mice is essential for optimal clearance of all five K. pneumoniae strains.

Extensive studies of immune defense against K. pneumoniae have been carried out with the 43816 laboratory strain, which is highly virulent in the mouse model. In mice infected with the 43816 strain, neutrophils were shown to be essential for optimal pulmonary clearance of bacteria, and the absence of IL-17, a cytokine that functions partially through facilitating neutrophil recruitment, markedly worsened infection (7, 14, 35). It is noteworthy that IL-17 also upregulates the expression of CCL2 and CCL7 and enhances macrophage recruitment in the lung (36, 37). CCL2 and CCL7 serve as ligands for CCR2 and are critical for monocyte migration and recruitment. Thus, it is possible that IL-17 contributes to pulmonary antimicrobial responses through monocyte-dependent pathways. Murine experimental studies have not focused on more recently isolated and increasingly antibiotic-resistant clinical strains of K. pneumoniae. Our results showed that the clinical strains were much more resistant to antibiotics but uniformly less pathogenic upon intratracheal inoculation of mice. Their clearance also relied on different host immune mechanisms. Although both neutrophils and Ly6C^hi CCR2⁺ monocytes were promptly recruited to the infected lung, their contributions to bacterial clearance varied for the different strains. Our results demonstrated that CCR2⁺ monocytes played an unexpectedly critical role in clearance of K. pneumoniae from the lung and recovery from infection. The microbiological and immunological mechanisms that underlie these differences are under investigation.

Neutrophils generally combat extracellular pathogens such as K. pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus. CCR2⁺ monocytes, on the other hand, have been implicated in defense against intracellular pathogens, including Listeria monocytogenes, Mycobacterium tuberculosis, and Toxoplasma gondii, although they also contribute to clearance of the fungal pathogen Aspergillus fumigatus (21, 23, 38, 39). As a small subset of blood circulating cells with remarkable plasticity, CCR2⁺ monocytes can directly participate in bacterial killing, transport live antigens, and modulate the function of other immune cells (40). The surprisingly important function of CCR2⁺ monocytes in defense against drug-resistant K. pneumoniae raises many interesting questions about their role in clearance of extracellular bacterial pathogens and will prompt further studies of their antimicrobial mechanisms and potential interactions with other effector cells.

Anti-Ly6G-mediated neutrophil depletion enabled us to deplete 93% of neutrophils in our experiments. Along similar lines, CXCR2^−/− mice have a 96% reduction of neutrophils in the lung (41), but in contrast to antibody-mediated depletion, in CXCR2^−/− mice trafficking is impaired and neutrophils are retained in the bone marrow (42, 43). These mice have also been shown to be highly susceptible to Gram-negative pneumonia (44).