International Journal of Medical Microbiology xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Medical Microbiology

journal homepage: www.elsevier.com/locate/ijmm

Caspase-11 deficiency impairs neutrophil recruitment and bacterial

clearance in the early stage of pulmonary Klebsiella pneumoniae infection
⁎
Jian Wanga,1, Yue Shaob,1, Wei Wangb, Shengjun Lic, Na Xinb, Fang Xiea, Chenghai Zhaob,
a
Department of Pathogen Biology, College of Basic Medical Science, China Medical University, Shenyang, China
b
Department of Pathophysiology, College of Basic Medical Science, China Medical University, Shenyang, China
c
Department of Immunology, College of Basic Medical Science, China Medical University, Shenyang, China

A R T I C L E I N F O A B S T R A C T

Keywords: Klebsiella pneumoniae (K. pneumoniae) is a gram-negative pathogen, and Klebsiella pneumonia is one of the most
Klebsiella pneumoniae common nosocomial infections. Canonical NLRP3 and NLRC4 inflammasome was found involved in innate
Caspase-11 immune response against K. pneumoniae, but the role of caspase-11 in K. pneumoniae infection remains un-
IL-1α defined. It was shown that Caspase-11 knockout blocked K. pneumoniae-induced IL-1α and IL-1β secretion and
IL-1β
pyroptosis of bone marrow-derived macrophages (BMDMs). Furthermore, caspase-11−/− mice exhibited im-
paired neutrophil recruitment and bacterial clearance in the early stage of K. pneumoniae infection, accompanied
by a reduction in IL-1α production. Moreover, IL-1α neutralizing antibody pretreatment was found to inhibit
neutrophil recruitment and bacterial clearance of wild-type mice. Together, these data suggest that caspase-11/
IL-1α pathway plays an important role in defending against K. pneumoniae by recruiting neutrophils in the early
stage of infection.

1. Introduction Klebsiella pneumoniae (K. pneumoniae) is a gram-negative pathogen,

Innate immune system plays an essential role in protection from
microorganism infection. Activation of canonical inflammasome is one
of the important immune responses to bacteria, viruses and other pa-
thogens. Assembly of canonical inflammasome involves some pattern
recognition receptors (PRRs), such as NOD-like receptor (NLR), which
recruits pro-caspase-1 with or without adaptor protein (Latz et al.,
2013; Man and Kanneganti, 2015; Sharma and Kanneganti, 2016).
Subsequently, pro-caspase-1 is auto-cleaved into active caspase-1, and
the latter further cleaves pro-IL-1β and pro-IL-18 to form mature IL-1β
and IL-18. Cytokine release as well as immune cell recruitment attri-
butes to inflammatory response and pathogen clearance.
In addition to canonical inflammasome, some gram-negative bac-
teria can activate pro-caspase-1 via LPS, which binds to another in-
flammatory caspase-caspase-11. Binding of caspase-11 with LPS simi-
larly cleaves pro-caspase-1, forming mature IL-1β and IL-18, therefore,
caspase-11 is also considered as a cytosolic PRR (Hagar et al., 2013;
Kayagaki et al., 2013; Shi et al., 2014). Activation of caspase-11 also
induces IL-1α release, and this process is not related to caspase-1.
Contrary to IL-1β, the role of IL-1α in innate immune response and
bacterial clearance has not been enough explored and elucidated.
which can lead to both community and nosocomial pneumonia, urinary
tract infection, liver abscess and sepsis, and Klebsiella pneumonia is one
of the most common nosocomial infections (Broberg et al., 2014). It has
been shown that canonical NLRP3 and NLRC4 inflammasome is in-
volved in innate immune response against K. pneumoniae (Cai et al.,
2012; Willingham et al., 2009). In the present study, caspase-11
knockout (caspase-11−/−) mice were infected with K. pneumoniae to
explore the role of caspase-11 in pulmonary K. pneumoniae infection.
2. Materials and methods
2.1. Animals
6–8-week-old specific pathogen free (SPF) caspase-11 knockout
(caspase-11−/−) mice and wild-type C57BL/6 (caspase-11+/+) mice
were used according to Guide to the Care and Use of Experimental
Animals (China). Experimental procedures were approved by China
Medical University Animal Care and Use Committee. Caspase-11−/−
mice (C57BL/6 background) were provided by the Jackson Laboratory,
and wild-type C57BL/6 mice were obtained from Animal Division
China Medical University.

⁎
Corresponding author.
E-mail address: zhaochenghai1@sina.com (C. Zhao).
1
These authors equally contributed to this work.

http://dx.doi.org/10.1016/j.ijmm.2017.09.012
Received 25 May 2017; Received in revised form 4 September 2017; Accepted 11 September 2017
1438-4221/ © 2017 Published by Elsevier GmbH.

Please cite this article as: Wang, J., International Journal of Medical Microbiology (2017), http://dx.doi.org/10.1016/j.ijmm.2017.09.012
J. Wang et al. International Journal of Medical Microbiology xxx (xxxx) xxx–xxx

Fig. 1. Activation of capase-11 by K. pneumoniae in caspase-11+/+ BMDMs

and mice. (A) K. pneumoniae 10031/13883 induced procaspase-11 and
caspase-11 expression in caspase-11+/+ BMDMs. (B) K. pneumoniae
10031/13883 induced procaspase-11 and caspase-11 expression in cas-
pase-11+/+ mice. All the experiments were performed three times. Each
group contained 5 mice.

Fig. 2. Blockade of K. pneumoniae-induced IL-1α and IL-1β secretion in caspase-11−/− BMDMs. (A, B) ELISA detection showed that caspase-11 knockout blocked K. pneumoniae 10031
−induced IL-1α and IL-1β secretion by BMDMs. (C) Lactate dehydrogenase assay revealed that caspase-11 knockout blocked K. pneumoniae 10031 –induced pyroptosis of BMDMs. (D, E)
ELISA detection showed that caspase-11 knockout blocked K. pneumoniae 13883 –induced IL-1α and IL-1β secretion by BMDMs. (F) Lactate dehydrogenase assay revealed that caspase-11
knockout blocked K. pneumoniae 13883 –induced pyroptosis of BMDMs. *P < 0.05, **P < 0.01, ***P < 0.001, vs casp-11+/+ 10031/13883(+). casp-11: caspase-11. Data are expressed
as mean ± SD, and all the experiments were performed three times.

2.2. Bone marrow-derived macrophage generation
Bone marrows were harvested from mouse femur and tibia. To
generate bone marrow-derived macrophages (BMDM), bone marrow
cells were plated in DMEM with 10% FBS and 10% M-CSF (L929 cell
supernatant).
2.3. Bacteria culture, inoculation and CFU measurement
K. pneumoniae (ATCC 10031, 13883) were grown overnight in TSB
at 37 °C. 2 × 107 K. pneumoniae in 20 μl saline were administrated to
anesthetized mice intranasally. At indicated time point, mice were sa-
crificed for examination. To quantify CFU (colony-forming units), sus-
pension fluid from BALF or homogenized lung tissue was inoculated in
sterile TSA at 37 °C for 24 h.
2.4. Neutralizing antibody treatment
IL-1α neutralizing antibody (Abcam, 50 μg or 100 μg per mice) or
IL-1β neutralizing antibody (Abcam, 50 μg or 100 μg per mice) was
used to treat caspase-11+/+ mice by peritoneal injection 2 h before K.
pneumoniae infection.
2.5. Bronchoalveolar lavage fluid collection and staining
1 ml saline was injected and withdrawn into the lungs to collect
bronchoalveolar lavage fluid (BALF), which was centrifuged at
2000 rpm for 5 min. The supernatants were collected for ELISA detec-
tion. After elimination of red blood cells, the precipitant was re-
suspended, and the suspension fluid was smeared. The number of
neutrophils was counted with Wright staining.
2.6. Western blot
Equal amount of protein was separated on 10% SDS-polyacrylamide
gels and transferred to PVDF membranes in a wet electron transfer
device. The membranes were blocked in 5% BSA in TBS containing
0.05% Tween 20 for 1 h at room temperature, and then incubated with

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Fig. 3. Impaired neutrophil recruitment and bacterial clearance in early stage of K. pneumoniae infection of caspase-11−/− mice. (A) Neutrophils in BALF of caspase-11−/− mice were less
than those of caspase-11+/+ mice. (B) Pulmonary MPO activity of caspase-11−/− mice was lower than that of caspase-11+/+ mice. (C, D) CFU in BALF and lung tissue of caspase-11−/−
mice were much more than those of caspase-11+/+ mice. *P < 0.05, **P < 0.01, ***P < 0.001, vs casp-11+/+. casp-11: caspase-11. Data are expressed as mean ± SD. Each group
contained 5 mice.

rabbit polyclonal antibody for caspase-11 (1:1000, Abcam) overnight at 11.3 × g of sample.
4 °C. After washed in TBS/Tween-20 three times, the membranes were
incubated in HRP-cinjugated goat anti-rabbit (1:5000) for 2 h at room
temperature. An ECL kit was used to visualize target proteins. 2.10. Hematoxylin-eosin staining

2.7. ELISA
Contents of IL-1α, IL-1β, IL-6 and TNF-α in BMDM supernatants and
bronchoalveolar lavage fluid (BALF) were detected by Mouse ELISA kits
(eBioscience) according to the manufacturer's instructions. The sensi-
tivity was 4 pg/ml for IL-1αand IL-6, and 8 pg/ml for IL-1β and TNF-α.
2.8. Cytotoxicity assay
Lactate dehydrogenase (LDH) assay kit (Beyotime® Biotechnology,
China) was used to evaluate the cytotoxicity of K. pneumoniae on
BMDM. 8 h after infection, cell supernatants were collected for analysis
of LDH release. A plate reader spectrophotomether was used to measure
the absorbance at 490 nm, and cytotoxicity values were calculated ac-
cording to the following equation: (ODsample − ODlowcontrol)/
(ODhighcontrol − ODlowcontrol) × 100.
2.9. Myeloperoxidase activity assay
Pulomonary myeloperoxidase (MPO) activity was detected by MPO
assay kit (Nanjing Jiancheng Corp., China) according to the manu-
facturer’s instructions. A plate reader spectrophotomether was used to
measure the absorbance at 460 nm. MPO (U/g lung tissue) value was
calculated using the following equation: (ODsample − ODcontrol)/
10% formalin was used to fix lung tissues, which were then em-
bedded in paraffin. 4 μm thick sections were stained with haematox-
ylin-eosin (HE), and observed using a Leica DMRB microscope. The
degree of pathology was judged blindly according to the following
criterion (Szarka et al., 1997): 0: no reaction in alveolar walls; 1: diffuse
reaction in alveolar walls, primarily neutrophilic, no thickening of al-
veolar walls; 2: diffuse presence of inflammatory cells in alveolar walls
with slight thickening; 3: distinct thickening of the alveolar walls due to
presence of inflammatory cells; 4: alveolar wall thickening with up to
25% of lung consolidated; 5: alveolar wall thickening with more than
50% of lung consolidated.
2.11. Statistical analysis
Student’s t-test was used to analyze the differences between two
groups. One way ANOVA was used to assess the effects of neutralizing
antibodies on infected caspase-11+/+ mice. Statistical analysis was
carried out by SPSS 13.0. A P-value less than 0.05 was considered
significant.

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Fig. 4. Suppression of pulmonary IL-1α production in early stage of K. pneumoniae infection of caspase-11−/− mice. (A) Level of IL-1α in caspase-11−/− mice were lower than that in
caspase-11+/+ mice. (B) Level of IL-1β was comparable between two types of mice infected with K. pneumoniae 10031, whereas slightly lower than that in caspase-11+/+ mice when these
mice were infected with K. pneumoniae 13883. (C, D) Levels of IL-6 and TNF-α in caspase-11−/− mice was higher than those in caspase-11+/+ mice. *P < 0.05, **P < 0.01,
***
P < 0.001, vs casp-11+/+. casp-11: caspase-11. Data are expressed as mean ± SD. Each group contained 5 mice.

3. Results 3.3. Caspase-11 deficiency impairs pulmonary neutrophil recruitment and

3.1. K. pneumoniae activates caspase-11 in caspase-11+/+ BMDMs and
mice
BMDMs from caspase-11+/+ mice were infected with K. pneumoniae
10031 or 13883 (MOI: 100) for 12 h. Procaspase-11 expression was
upregulated by both strains, moreover, cleaved caspase-11 expression
was also induced by these bacteria (Fig. 1A). 6 h after infection with
2 × 107K. pneumoniae 10031 or 13883, lungs of caspase-11+/+ mice
expressed more procaspase-11 and cleaved caspase-11, compared with
uninfected mice (Fig. 1B). These data indicate that K. pneumoniae can
induce caspase-11 production and activation.
bacterial clearance in early stage of K. pneumoniae infection
Both caspase-11+/+ and caspase-11−/− mice were infected with K.
pneumoniae 10031 for 6 h. Neutrophils in bronchoalveolar lavage fluid
(BALF) of caspase-11−/− mice were less than those of caspase-11+/+
mice (Fig. 3A). Consistently, pulmonary MPO activity of caspase-11−/−
mice was lower than that of caspase-11+/+ mice (Fig. 3B). CFU in BALF
and lung tissue of caspase-11−/− mice were much more than those of
caspase-11+/+ mice (Fig. 3C and D). Similar results were found when
caspase-11+/+ and caspase-11−/− mice were infected with K. pneu-
moniae 13883 (Fig. 3A, B, C and D).

3.4. Caspase-11 deficiency suppressed pulmonary IL-1α production in early

3.2. Caspase-11 deficiency blocks K. pneumoniae-induced IL-1α and IL-1β
secretion in BMDMs
+/+ −/−
BMDMs from caspase-11 and caspase-11 mice were infected
with K. pneumoniae 10031 (MOI: 100) for 12 h. Secretion of both IL-1α
and IL-1β was upregulated in caspase-11+/+ BMDMs (Fig. 2A and B).
However, induction of IL-1α was completely blocked, and induction of
IL-1β secretion was partially blocked, when caspase-11 was knockout
(Fig. 2A and B). Moreover, cytotoxicity of K. pneumoniae 10031 on
caspase-11−/− BMDMs was significantly lower than that on caspase-
11+/+ BMDMs (Fig. 12 Fig. 12C). Similar results were observed when
BMDMs from caspase-11+/+ and caspase-11−/− mice were infected
with K. pneumoniae 13883 (Fig. 2D, E and F). These data indicate that
caspase-11 mediates K. pneumoniae-induced IL-1α and IL-1β secretion,
and BMDM pyroptosis.
stage of K. pneumoniae infection
Both caspase-11+/+ and caspase-11−/− mice were infected with K.
pneumoniae 10031 for 6 h. ELISA was used to evaluate cytokine pro-
duction in BALF. Level of IL-1α in caspase-11−/− mice was lower than
that in caspase-11+/+ mice, and level of IL-1β was comparable between
two types of mice, whereas levels of IL-6 and TNF-α in caspase-11−/−
mice were higher than those in caspase-11+/+ mice (Fig. 4A, B, C and
D). Similar secretion pattern of IL-1α, IL-6 and TNF-α was observed,
except that level of IL-1β in caspase-11−/− mice was slightly lower than
that in caspase-11+/+ mice, when these mice were infected with K.
pneumoniae 13883 (Fig. 4A, B, C and D).

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Fig. 5. Inhibition of neutrophil recruitment and bacterial clearance in early stage of K. pneumoniae infection of caspase-11+/+ mice by IL-1α neutralizing antibody. (A) IL-1α neutralizing
antibody reduced IL-1α in BALF. (B) IL-1β neutralizing antibody reduced IL-1β in BALF. (C) IL-1α neutralizing antibody reduced neutrophils in BALF. (D) IL-1α neutralizing antibody
suppressed MPO activity of lung tissue. (E, F) IL-1α neutralizing antibody increased CFU in BALF and lung tissue. *P < 0.05, **P < 0.01, ***P < 0.001. NS: normal saline. Data are
expressed as mean ± SD. Each group contained 5 mice.

3.5. IL-1α blockade inhibits pulmonary neutrophil recruitment and bacterial
clearance in early stage of K. pneumoniae infection of caspase-11+/+ mice
To explore the role of IL-1α and IL-1β in pulmonary neutrophil
recruitment and K. pneumoniae clearance in early stage of infection,
caspase-11+/+ mice infected with K. pneumoniae 10031 were pretreated
with IL-1α neutralizing antibody or IL-1β neutralizing antibody. IL-1α
neutralizing antibody and IL-1β neutralizing antibody significantly
reduced IL-1α and IL-1β in BALF, respectively (Fig. 5A and B). IL-1α
neutralizing antibody reduced neutrophils in BALF, and MPO activity of
lung tissue (Fig. 5C and D), and increased CFU in BALF and lung tissue
(Fig. 5E and F). IL-1β neutralizing antibody had no significant effect on
neutrophils in BALF, MPO activity of lung tissue, and CFU in BALF and
lung tissue (Fig. 5C, D, E and F).

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Fig. 6. Exacerbation of pulmonary pathological changes in the persistent stage of K. pneumoniae infection of caspase-11−/− mice. (A, B) No obvious pathological change was observed in
lung tissue without K. pneumoniae infection. (C, D) 24 h after infection with K. pneumoniae 10031, more neutrophils infiltrated and alveolar wall thickened more distinctly in caspase-11−/
−
mice. (E-F) 72 h after infection, caspase-11−/− mice displayed more severe bronchopneumonia characterized by extensive neutrophil infiltration and lung consolidation. *P < 0.05, vs
casp-11+/+. casp-11: caspase-11. Uninf: uninfection. Data are expressed as mean ± SD. Each group contained 5 mice, and only one representative image was shown.

3.6. Caspase-11 deficiency exacerbates pulmonary pathological changes in
the persistent stage of K. pneumoniae infection
No obvious pathological change was observed in lung tissue from
caspase-11+/+ and caspase-11−/− mice without K. pneumoniae infec-
tion (Fig. 6A and B). 24 h after infection with K. pneumoniae 10031,
pulmonary pathology score of caspase-11−/− mice was significantly
higher than that of caspase-11+/+ mice; more neutrophils infiltrated in
alveolar space and bronchial lumen, and alveolar wall thickened more
distinctly in caspase-11−/− mice, compared with caspase-11+/+ mice
(Fig. 6C and D). 72 h after infection, caspase-11−/− mice displayed
more severe bronchopneumonia characterized by a large number of
neutrophils infiltrating in the lumen of bronchi and large bronchioles,
and many areas of the lungs were highly consolidated (Fig. 6E and F).
4. Discussion
Our study showed that K. pneumoniae-induced IL-1α and IL-1β se-
cretion, and macrophage pyroptosis were abrogated by caspase-11
knockout, indicating that K. pneumoniae can activate caspase-11
pathway. Caspase-11 is mainly expressed in monocytes and epithelial
cells (Shi et al., 2014). Activation of caspase-11 initiates non-canonical
inflammasome pathway, which induces caspase-1 activation in-
dependent of canonical inflammasome receptors such as NLR, AIM2
and IFI16. Some gram-negative bacteria including Escherichia coli, Sal-
monella typhimurium and Shigella flexneri can activate caspase-11, and
LPS entering into the cytosol is responsible for this activation (Kayagaki
et al., 2013). Moreover, reactive oxygen species (ROS) induced by
bacterial infection was reported to be involved in caspase-11 activation
via JNK (Lupfer et al., 2014).
Studies using caspase-11−/− mice have revealed that activation of
caspase-11 plays a protective role in some gram-negative bacterial in-
fection. Caspase-11 deficiency made the mice more susceptible to le-
gionella pneumophila, Escherichia coli, Salmonella typhimurium infection
(Akhter et al., 2012; Knodler et al., 2014). Our study showed that
caspase-11 deficiency impeded neutrophil recruitment in the early
stage of K. pneumoniae infection, accompanied by impaired bacterial
clearance as well as exaggerated pulmonary pathological changes,
therefore, indicating that caspase-11 plays an important role in de-
fending against K. pneumoniae.
Activation of caspase-11 induces secretion of not only IL-1β but also
IL-1α, and IL-1α induction is not dependent on caspase-1. The role of
IL-1α in caspase-11 pathway has not been fully elucidated. Some recent
studies demonstrated that IL-1α induced neutrophil recruitment to fa-
cilitate bacterial clearance (Al Moussawi and Kazmierczak, 2014;
Casson et al., 2013). It was found that LPS could induce macrophage
death and IL-1α release (Dagvadorj et al., 2015). IL-1α was also re-
vealed responsible for neutrophil recruitment in infection with some
fungi and viruses (Caffrey et al., 2015; Di Paolo et al., 2014; Milora
et al., 2014). Moreover, IL-1α was observed to be involved in neu-
trophil recruitment in some sterile inflammation (Scarpa et al., 2015;
Tynan et al., 2014).
Our study revealed that caspase-11 deficiency resulted in a reduc-
tion in IL-1α production in the early stage of K. pneumoniae infection,
and IL-1α neutralizing antibody pretreatment suppressed neutrophil
recruitment and bacterial clearance of wild-type mice. These data
suggest that IL-1α reduction may attribute to impaired neutrophil re-
cruitment and bacterial clearance of caspase-11−/− mice. IL-1α-in-
duced neutrophil recruitment may be related to chemokine IL-8.
Actually, some studies have confirmed that IL-1α can induce IL-8 ex-
pression (Cheng et al., 2008; Dongari-Bagtzoglou and Kashleva, 2003;
Orjalo et al., 2009). One in vitro study recently showed that IL-1α

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induced IL-8 in bronchial epithelial cells (Bellon et al., 2017).
In summary, caspase-11 deficiency impaired neutrophil recruitment
in the early stage of K. pneumoniae infection, resulting in reduced
bacterial clearance and exacerbated pulmonary pathological changes;
caspase-11 deficiency inhibited IL-1α secretion which was observed
implicated in neutrophil recruitment of caspase-11+/+ mice infected
with K. pneumoniae. These data suggest that caspase-11 can sense and
respond to K. pneumoniae by recruiting neutrophils via IL-1α.
Acknowledgments
For financial support, we acknowledge the Natural Scientific
Foundation of China (81502107).
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