Nlrp3 inflammasome pathway has a critical role in the host immunity against clinically relevant acinetobacter baumannii pulmonary infection

ABSTRACT The opportunistic Gram-negative bacterium _Acinetobacter baumannii_ (AB) is a leading cause of life-threatening nosocomial pneumonia. Outbreaks of multidrug resistant (MDR)-AB

belonging to international clones (ICs) I and II with limited treatment options are major global health threats. However, the pathogenesis mechanisms of various AB clonal groups are

understudied. Although inflammation-associated interleukin-1β (IL-1β) levels and IL-1 receptor antagonist polymorphisms were previously implicated in MDR-AB-related pneumonia in patients,

whether inflammasomes has any role in the host defense and/or pathogenesis of clinically relevant _A. baumannii_ infection is unknown. Using a sublethal mouse pneumonia model, we demonstrate

that an extensively drug-resistant clinical isolate (ICII) of _A. baumannii_ exhibits reduced/delayed early pulmonary neutrophil recruitment, higher lung persistence, and, most importantly,

elicits enhanced IL-1β/IL-18 production and lung damage through NLRP3 inflammasome, in comparison with _A. baumannii_-type strain. _A. baumannii_ infection-induced IL-1β/IL-18 production is

entirely dependent on NLRP3-ASC-caspase-1/caspase-11 pathway. Using Nlrp3−/− mice infection models, we further show that while NLRP3 inflammasome pathway contributes to host defense against

_A. baumannii_ clinical isolate, it is dispensable for protection against _A. baumannii_-type strain. Our study reveals a novel differential role for NLRP3 inflammasome pathway in the

immunity against clinically relevant _A. baumannii_ infections, and highlights inflammasome pathway as a potential immunomodulatory target. SIMILAR CONTENT BEING VIEWED BY OTHERS INTERFERON

SIGNALLING AND NON-CANONICAL INFLAMMASOME ACTIVATION PROMOTE HOST PROTECTION AGAINST MULTIDRUG-RESISTANT _ACINETOBACTER BAUMANNII_ Article Open access 12 November 2024 IMPAIRMENT IN

INFLAMMASOME SIGNALING BY THE CHRONIC _PSEUDOMONAS AERUGINOSA_ ISOLATES FROM CYSTIC FIBROSIS PATIENTS RESULTS IN AN INCREASE IN INFLAMMATORY RESPONSE Article Open access 04 March 2021 THE

PRO-INFLAMMATORY EFFECT OF STAPHYLOKINASE CONTRIBUTES TO COMMUNITY-ASSOCIATED _STAPHYLOCOCCUS AUREUS_ PNEUMONIA Article Open access 23 June 2022 INTRODUCTION _Acinetobacter baumannii_ has

recently emerged as one of the most successful Gram-negative bacteria causing both nosocomial and community-acquired infections globally.1, 2 Although hospital-acquired pneumonia constitutes

the most prevalent clinical manifestation associated with _A. baumannii_ (AB), sepsis, urinary tract infection, meningitis, endocarditis, skin, and soft tissue infections are also common.3

Mortality rates of ventilator-associated and community-acquired pneumonia caused by _A. baumannii_ ranges from 40 to 70%.4, 5 The clinical success of _A. baumannii_ is attributed to its

ability to (i) form biofilms and resist environmental stresses such as desiccation and disinfectants, (ii) develop anti-microbial resistance, (iii) acquire genetic material by horizontal

gene transfer from unrelated genera, and (iv) adhere, colonize, and invade mammalian cells.6 In fact, because of its remarkable ability to develop resistance to nearly all known antibiotics,

_A. baumannii_ has recently been classified as a “red alert” human pathogen.7 This highlights the importance of developing new/improved or alternative strategies to combat _A. baumannii_.

Based on its virulence and ability to cause epidemics, _A. baumannii_ is classified into three predominant clones (international clones (ICs) I, II, and III). Multidrug-resistant and

carbapenem (the last resort of antibiotics to treat _A. baumannii_)-resistant virulent _A. baumannii_ belonging to ICs I and II are the most prevalent clinically relevant _A. baumannii_

strains causing outbreaks worldwide.1, 8, 9 ICII is the most successful clone of _A. baumannii_ causing outbreaks in hospital environments worldwide, especially in Asia. However, what

determines the clinical success of ICI and ICII clones is not well known. A thorough understanding of the host–pathogen interaction is critical for designing new therapeutics and vaccines.

However, currently only limited information is available on the virulence mechanisms and associated protective host immune response, especially during virulent clinically relevant _A.

baumannii_ infections. Development of pneumonia results from a complex interplay between the pathogen and the pulmonary mucosal immunity. Host innate immune system is critical for

controlling _A. baumannii_ infection, and may also have significant roles in immunopathogeneis associated with _A. baumannii_ infection. The acute rapid infection kinetics of _A. baumannii_

infections also further highlights the importance of innate immunity in controlling _A. baumannii_. Individuals with suppressed immune status are particularly vulnerable to _A. baumannii_

infections.10 Immunomodulatory therapies in patients with compromised immune system could offer an attractive strategy to tackle _A. baumannii_ infections. However, a thorough understanding

of host immune response to _A. baumannii_ infection is required to tackle effectively the infectious threat of _A. baumannii_ infection. Previous studies have identified some of the innate

immune mechanisms involved in host response to _A. baumannii_ infection. Neutrophils and Toll-like receptor 4 (TLR4) pathway have important roles in the pulmonary innate immune defense

against _A. baumannii_ infection.11, 12, 13, 14, 15 However, TLR2 pathway has either a protective or pathological role during pulmonary _A. baumannii_ infection.16 The role of intracellular

innate immune receptors in _A. baumannii_ immunity is only beginning to be appreciated. A role for intracellular NOD1/2 pathway in the innate immune response to _A. baumannii_ infection was

identified previously.17 A recent report has also demonstrated the role of the intracellular receptor TLR9 in the pulmonary host defense against _A. baumannii_.18 All these previous studies

used either sensitive strains (not clinically relevant drug-resistant forms) of _A. baumannii_ or used mouse sepsis models of infection and hence may not accurately depict the interaction of

clinically relevant _A. baumannii_ with the pulmonary innate immune system. Using mouse pneumonia infection models, previous studies have shown that clinically relevant more virulent forms

of _A. baumannii_ (belonging to ICI and II) survive and persist in the lungs of the host by inducing only a weak innate immune inflammatory response in comparison with less/nonvirulent

forms.19, 20 This implies that drug-resistant virulent _A. baumannii_ may evade/suppress the innate immune response to persist and cause various pathological consequences in the host.

However, the underlying mechanism or the innate immune pathways involved in this are not fully characterized yet. Inflammasomes are important intracellular multiprotein innate immune

complexes that are implicated in infection control, inflammation, and tissue injury. An earlier study conducted in patients with _A. baumannii_-associated pneumonia has showed that higher

concentration of interleukin-1β (IL-1β) (a marker of inflammasome activation) observed in the bronchoalveolar lavage (BAL) of patients correlated with the higher bacterial burden or the

presence of virulent bacteria in their alveoli.21 This study has proposed that levels of BAL IL-1β could be considered as a marker for ongoing inflammation in response to persistent

bacterial infection in the lung. Yet, another clinical study showed that a polymorphism in the gene encoding the anti-inflammatory antagonist of IL-1 receptor (IL-1RA) (a competitive

inhibitor of IL-1β) is associated with the risk of developing multidrug-resistant _A. baumannii_-related pneumonia.22 These studies clearly indicated an involvement of IL-1-associated

pathways in the pulmonary immune response to clinically relevant _A. baumannii_ infection. However, the role of inflammasome in the host immune response to clinically important _A.

baumannii_ is not characterized. Inflammasomes sense either the microbial stimuli or danger signals and regulates two major host protective responses: (a) secretion of highly proinflammatory

cytokines IL-1β and IL-18 and (b) induction of pyroptosis.23 The best-characterized inflammasome is the NLRP3 inflammasome. NLRP3 has been implicated in various pulmonary diseases including

acute respiratory distress syndrome, sepsis, and several pulmonary bacterial and viral infections including _Klebsiella pneumoniae_, _Streptococcus pneumoniae_, _Staphylococcus aureus_,

_Chlamydia pneumoniae_, _Mycobacterium tuberculosis_, _Legionella pneumophila_, influenza virus, human rhinovirus, respiratory syncytial virus, and _Aspergillus fumigatus_ (for a review see

Chaput _et al._24). In addition, differential activation of NLRP3 inflammasome by bacterial pathogens of varying virulence resulted in different pathological outcomes.25 However, (i) whether

NLRP3 inflammasome pathway has any role in the host defense to _A. baumannii_ infection and (ii) whether there is any differential interaction of virulent _A. baumannii_ with NLRP3 that can

impact the clinical outcomes associated with _A. baumannii_ infections is also currently not known. In this report, we investigated the hypothesis that clinically relevant

antibiotic-resistant forms of _A. baumannii_ belonging to ICI and ICII may interact differently with the inflammasome pathway leading to host defense and also pathological outcomes.

Particularly, we aimed to understand the interaction of _A. baumannii_ with the pulmonary NLRP3 inflammasome system. For this study, we used an extensively drug-resistant (XDR) _A.

baumannii_ isolate (AB-8879, belonging to ICII clonal group) that caused severe sepsis and bacteremia in patients of the burns intensive care unit.26, 27 AB-8879 strain belongs to

carbapenem-resistant _A. baumannii_, and in addition, it shows polymyxin B heteroresistance. The results of our study revealed that the clinical _A. baumannii_ isolate caused a predominant

activation of NLRP3 inflammasome _in vivo_, and that NLRP3 was important during host immune response to _A. baumannii_ infection. RESULTS _A. BAUMANNII_ INFECTION PRIMES AND ACTIVATES THE

INFLAMMASOME COMPLEX IN MOUSE BONE MARROW-DERIVED MACROPHAGES IL-1β levels/IL-1RA polymorphism are previously reported to be associated with multidrug-resistant-AB-related pneumonia in

patients21, 22, indicating that inflammasome activation could have a role in clinically relevant _A. baumannii_ infections. Hence, we aimed to investigate the role of inflammasomes during

clinically relevant _A. baumannii_ (belonging to ICI and II implicated in outbreaks worldwide) infections. An XDR clinical isolate of _A. baumannii_ causing severe sepsis and bacteremia in

patients in burns intensive care unit from Singapore belonging to the ICII (carbapenem-resistant (8879, ICII))26, 27 was used for our studies. We first ascertained whether _A. baumannii_ can

activate the inflammasome pathway in macrophages. Bone marrow-derived macrophages (BMDMs) were infected with AB-8879. Transcript levels of procaspase-1 and pro-IL-1β were determined by

quantitative reverse transcription-PCR. _A. baumannii_ infection led to 5.5-fold increase in procaspase-1 mRNA (_P_=0.004) (Figure 1a) and 344-fold increase in pro-IL-1β mRNA (_P_=0.003)

levels (Figure 1b) at 24 h after infection. Next, to functionally verify whether inflammasome complex is also activated at the protein level, we first determined the levels of activated

caspase-1 by western blot analysis. _A. baumannii_ infection resulted in the cleavage of procaspase-1 to its active form caspase-1 p20 (Figure 1c). We next examined whether the caspase-1

activation observed during _A. baumannii_ infection further leads to the generation of IL-1β and IL-18, by measuring these cytokines in the infected BMDM culture supernatants. The release of

IL-1β (Figure 1d) and IL-18 (Figure 1e) was found to be induced upon _A. baumannii_ infection of lipopolysaccharide (LPS)-primed BMDMs. LPS+ATP-treated cells were used as positive controls.

_A. baumannii_ was also able to prime the inflammasome as it mediated IL-1β (Figure 1f) and IL-18 (Figure 1g) release in the absence of external LPS prestimulation. These results clearly

demonstrate that _A. baumannii_ is capable of priming as well as activating the inflammasome complex. _A. BAUMANNII_ INDUCES IL-1Β AND IL-18 PRODUCTION THROUGH NLRP3 INFLAMMASOME NLRP3 and

NLRC4 are major inflammasome complexes that can independently sense bacterial pathogens and lead to the production of IL-1β and IL-18 cytokines. NLRC4 complex is activated by bacterial

flagellin; however, the bacterial ligands for NLRP3 inflammasome are still unknown. As _A. baumannii_ is a non-flagellated bacterium, we hypothesized that the infection-induced IL-1β/IL-18

production would be likely through the activation of NLRP3 pathway. We first determined the expression of NLRP3 at the mRNA level and found that it was upregulated by 3.5-fold (_P_=0.006)

upon _A. baumannii_ infection (Figure 2a). Next, we infected BMDMs from wild-type C57BL/6 mice (WT) or Nlrp3-knockout (Nlrp3−/−) mice and measured the production of catalytically active

caspase-1 by western blot. While the BMDMs from WT mice produced mature (active) caspase-1, Nlrp3−/− macrophages failed to generate active caspase-1 following _A. baumannii_ infection

(Figure 2b). As caspase-1 processing is fully dependent on Nlrp3, a putative role of Nlrc4 in _A. baumannii_ infection was excluded. In accordance with the lack of caspase-1 activation,

IL-1β and IL-18 (Figure 2c,d) production was also completely abrogated in Nlrp3−/− macrophages upon _A. baumannii_ infection. The production of MIP-2 (Figure 2e) and tumor necrosis factor-α

(Figure 2f) were unaffected by the absence of NLRP3, indicating that the absence of NLRP3 does not lead to a generalized defect in inflammatory cytokine production and that NLRP3 is

specifically involved in the synthesis of inflammatory IL-1β and IL-18 cytokines. To rule out the possibility that the reduced levels of IL-1β/IL-18 in Nlrp3−/− macrophages is due to reduced

intracellular invasion of bacteria, the intracellular bacterial load in BMDM was ascertained at 4 h after infection. As seen in Supplementary Figure S1a online, there was no significant

difference in the intracellular bacterial load between WT and Nlrp3−/− macrophages. NLRP3 inflammasome is activated by various DAMPs such as ROS, cathespin B, and potassium ion efflux.28 We

wanted to explore which of these mediators (if any) are involved in activating the inflammasome pathway during _A. baumannii_ infection. We first explored if cellular invasion of bacteria is

required to generate IL-1β through the above-mentioned mechanisms. For this, BMDMs were treated with cytochalasin D, which inhibits actin polymerization and therefore phagocytosis of

bacteria. IL-1β release was found to be inhibited by 1.6-fold by cytochalasin D treatment (Figure 2g). Heat-killed bacteria also elicited 1.7-fold lesser IL-1β. As ROS has been documented in

the control of _A. baumannii_,14, 29 we next treated the cells with ROS inhibitor APDC to see whether ROS are involved in the generation of IL-1β. We found that APDC treatment inhibited _A.

baumannii_-induced IL-1β release by 1.7-folds (Figure 2g). Treatment with KCl, a potassium efflux inhibitor, reduced the IL-1β release by 1.6-folds (Figure 2g). Cathepsin B is a potential

NLRP3 activator, which is released upon lysosomal damage. Pharmacological inhibition of cathepsin B by Ca-074-Me also inhibited IL-1β release by 1.5-fold (Figure 2g). Since blocking none of

the tested DAMPs fully abrogated IL-1β release, we conclude that each of the DAMPs evaluated may contribute only partially to _A. baumannii_-induced inflammasome activation. Therefore, these

results show that upon _A. baumannii_ infection, macrophages release various DAMPs, which may contribute to NLRP3 inflammasome activation. Having found the role of NLRP3 pathway during _A.

baumannii_ infection, we next characterized the downstream signaling components of NLRP3 pathway that is involved in the caspase-1 activation/IL-1β generation during _A. baumannii_

infection. As ASC is the adaptor protein and a major downstream component of NLRP3 inflammasome, we further assessed whether ASC is also involved in caspase-1 activation during _A.

baumannii_ infection. Infected Asc−/− macrophages failed to generate active caspase-1 p20, implying that ASC is required for caspase-1 activation during _A. baumannii_ infection (Figure 3a).

NLRP3/ASC complex ultimately recruits Caspase-1 to the inflammasome. Caspase-1 cleaves pro-IL-1β and pro-IL-18 to their active forms either independently or under the regulation of

Caspase-11.30 _A. baumannii_ failed to activate Caspase-1 in Casp-11−/− (single knockout) macrophages (Figure 3a). Casp-1−/− macrophages were used as positive controls. Macrophages from

Asc−/−, Casp-1−/−, and Casp-11−/− single-knockout or Casp-1/11−/− double-knockout mice failed to generate IL-1β (Figure 3b) or IL-18 (Figure 3c) cytokines upon _A. baumannii_ infection.

However, the induction of transcripts of IL-1β and IL-18 was not affected by the absence of Nlrp3, Asc, Caspase-1, or Caspase-11 (Supplementary Figure S1b,c, respectively). Thus, we

conclusively prove that the production of IL-1β and IL-18 during _A. baumannii_ infection is driven by both canonical and noncanonical NLRP3 inflammasome pathway, and also suggests that

NLRP3 inflammasome pathway may have a role in the host immunity to _A. baumannii_ infections. CLINICAL ISOLATE OF _A. BAUMANNII_ ELICITS ENHANCED NLRP3-DEPENDENT PRODUCTION OF IL-1Β AND

IL-18 AND PYROPTOSIS IN THE LUNGS IN COMPARISON WITH THE TYPE STRAIN _IN VIVO_ We next assessed the _in vivo_ role of NLRP3 inflammasome during _A. baumannii_ pneumonia by the clinical

isolate and also examined if inflammasome activation is a common feature of all _A. baumannii_ strains. We used AB-8879 isolate (as a representative of clinically relevant _A. baumannii_),

and for the comparison purpose, we used the type strain 19606 (an antibiotic-susceptible strain that has been used extensively for pathogenesis studies before) and compared their ability to

activate inflammasome pathway using a mouse pneumonia model. As our aim was to study host immune response to _A. baumannii_ pneumonia infection, we used a sublethal (self-limiting) pneumonia

model using immunocompetent mice. Based on previous published reports from other research groups5, 15 and our unpublished data, a sublethal (5 × 107 CFU) mouse pneumonia model for _A.

baumannii_ infection in C57BL/6 mice was used for the studies. C57BL/6 mice were intranasally infected with 5 × 107 CFU of _A. baumannii_ using either AB-8879 or AB-19606, and the levels of

inflammasome-associated cytokines IL-1β and IL-18 was assessed in the BAL fluid at 4 and 24 h after infection. Only low levels of IL-1β were detected in the BAL of either AB-8879- or

AB-19606-infected mice at 4 h after infection (Figure 4a). Moreover, the levels of IL-1β were comparable between the strains at this time point. Similarly, although both AB-8879 and AB-19606

produced measurable amounts of IL-18 at 4 h after infection, the levels were comparable between the strains (Figure 4b). However at 24 h after infection, AB-8879 elicited robust production

of both IL-1β and IL-18 cytokines (Figure 4a,b). However, interestingly, these cytokines were produced only in significantly lesser amounts by AB-19606 strain as compared with AB-8879 (IL-1β

was 3.5-fold lesser, _P_=0.03, IL-18 was 3-fold lesser, _P_=0.04) (Figure 4a,b). Intriguingly, both AB-8879 and AB-19606 elicited comparable levels of both IL-1β and IL-18 during _ex vivo_

stimulation of mouse BMDMs (Supplementary Figure S2a,b, respectively). Moreover, a dose–response analysis also showed that comparable levels of IL-1β was produced _ex vivo_ by AB-19606 and

AB-8879 at all the multiplicity of infections (MOIs) and at both time points tested (4 and 24 h after infection) (Supplementary Figure S2c,d, respectively). These experiments indicate that

AB-8879 may differentially interact with the host immune system _in vivo_, thereby leading to the enhanced inflammasome activation. Additionally, AB-8879 induced higher cell death/lung

damage as evidenced by higher lactate dehydrogenase (LDH) levels31 in the BAL from AB-8879-infected mice in comparison with AB-19606-infected mice (1.5-fold difference, _P_=0.04) (Figure

4c). Cell death can also be mediated by caspase-11-mediated pathway that is associated with the secretion of IL-1α, a related cytokine to IL-1β.32 Notably, we also observed higher levels of

IL-1α in the BAL of AB-8879-infected mice in comparison with mice infected with AB-19606 (Supplementary Figure S3). These results clearly demonstrate that AB-8879 induces robust inflammasome

activation and cell death _in vivo_. Similar to our _in vitro_ results, our _in vivo_ infection experiments using Nlrp3−/− mice showed that the IL-1β and IL-18 production by AB-8879 was

completely dependent on NLRP3 inflammasome (Figure 4d,e). Additionally, cell death/lung damage was significantly reduced in Nlrp3−/− mice (1.5-fold lesser LDH (_P_=0.04) was detected in BAL

of Nlrp3−/− mice; Figure 4f). Thus, NLRP3 inflammasome pathway contributes to IL-1β/IL-18 production, and cell death/lung damage associated with clinically relevant AB-8879 infection. We

next explored the mechanism behind the enhanced ability of AB-8879 to activate inflammasome pathway _in vivo_. We observed that although there was no significant difference in the bacterial

load between the AB-19606 and AB-8879 at early time points (4 h), the mice infected with the AB-8879 had significantly higher bacterial load in the lungs at all the late time points tested

(24, 48, and 72 h after infection) (Figure 5a). AB-8879 persisted in the lungs even at 72 h after infection; however, all the mice cleared the infection by 96 h. In addition, AB-8879

successfully disseminated to extrapulmonary organs (such as the blood and spleen) at all the tested time points (Figure 5b,c, respectively). In contrast, we found that as per previous

published reports, the type strain AB-19606 was completely cleared from the lungs by ∼48 h after infection, and there was minimal or no extra pulmonary dissemination (Figure 5a). Consistent

with the higher bacterial load and delayed bacterial clearance, we also observed significant weight loss in the mice infected with the AB-8879 strain. The higher lung bacterial burden

observed in mice infected with AB-8879 could be one of the potential factors contributing to the enhanced activation of NLRP3 inflammasome pathway by AB-8879 in comparison to AB-19606. It is

interesting to note that there was no significant difference in the growth rate of AB-8879 in comparison with AB-19606 _in vitro_.33 This ruled out the possibility that higher intrinsic

growth rate of AB-8879 accounts for its increased load _in vivo_ compared to AB-19606, but rather points towards the possibility of an immune evasion mechanism by the clinical isolate. _A.

BAUMANNII_ CLINICAL ISOLATE EXHIBITS REDUCED EARLY NEUTROPHIL INFLUX IN THE LUNGS IN COMPARISON TO THE TYPE STRAIN Since clinical isolate of _A. baumannii_ attained higher bacterial load

compared with type strain _in vivo_, we hypothesized that virulent _A. baumannii_ potentially delays/evades/inhibits some of the early innate immune responses, leading to increased bacterial

load during the early course of infection, and thereby resulting in enhanced activation of inflammasome pathway. We further reasoned that AB-19606 is effectively controlled by innate immune

control mechanisms in the early stages of infection itself, resulting in diminished inflammasome activation. Early recruitment of neutrophils has a critical role in host resistance to

respiratory _A. baumannii_ infection.11, 13, 14, 15 Therefore, we first sought to determine if there is any difference in the lung/BAL neutrophil recruitment kinetics of AB-8879 and

AB-19606. Neutrophil numbers were determined by immunostaining for myeloperoxidase (MPO). We found that the mice infected with AB-8879 had 2.5-fold lesser MPO-positive cells in the lungs as

compared with AB-19606-infected mice at 4 h after infection (_P_=0.03) (Figure 6a and Supplementary Figure S4a). Similarly, 2.3-fold lesser MPO-positive cells was observed in the BAL of mice

infected with AB-8879 as compared with AB-19606 at 4 h after infection (_P_=0.04) (Figure 6b). However, no difference in MPO-positive cells in lungs/BAL was observed among mice infected

with either of the two strains at 24 h after infection (Figure 6a and Supplementary Figure S4b for the lungs and Figure 6b for the BAL, respectively). Further experiments were carried out to

confirm the differences in the neutrophil numbers observed at 4 h after infection. MPO levels in lungs were also determined by enzyme-linked immunosorbent assay (ELISA) and were found to be

1.3-fold lesser in mice infected with AB-8879 as compared with mice infected with AB-19606 (_P_=0.01) (Figure 6c). Neutrophil elastase activity was found to be 2.4-fold lesser in BAL of

mice infected with AB-8879 than in the BAL from mice infected with AB-19606 (_P_=0.02) (Figure 6d). Furthermore, Giemsa staining of the BAL at 4 h after infection also showed that

AB-8879-infected mice had twofold lesser number of neutrophils as compared with AB-19606 (_P_=0.005) (Figure 6e). Consistent with this, there was significantly lesser number of total immune

cells in the BAL of mice infected with AB-8879 in comparison with that from AB-19606 (Figure 6e). However, the percentage of alveolar macrophages was comparable in AB-19606 and AB-8879 at

this time point (Figure 6e). The percentage of other immune cells was negligible. We also performed FACS analysis of the BAL fluid for neutrophil enumeration (Figure 6f). The results show

that there were significantly lesser Gr-1/CD11b-positive neutrophils (∼1.8-fold) in the BAL of mice infected with AB-8879 in comparison with AB-19606. Collectively, these results clearly

demonstrate that AB-8879 recruits lesser neutrophils to the lungs early on during infection in comparison with AB-19606. The observed early reduction in the number of neutrophil/delay in

early neutrophil recruitment to the lungs could thus potentially account for the higher bacterial load and subsequent enhanced inflammasome activation exhibited by the clinical isolate

AB-8879. Chemoattractants such as GROα (KC or CXCL1), MIP-2 (CXCL2), and CXCL5 are important mediators of neutrophil recruitment to the lungs during infection.12, 34, 35, 36, 37 Thus, we

next assessed whether the presence of lesser neutrophil numbers in the lungs of mice infected with AB-8879 in comparison with AB-19606 (during early time points of infection) is due to their

differential ability to elicit neutrophil chemoattractant production. However, we found that there was no impairment in the production of any of the tested chemokines (GROα, MIP-2, and

CXCL5) in the BAL of AB-8879 in comparison with AB-19606 both at 4 and 24 h after infection (Supplementary Figure S5a–c, respectively). IL-12 is one of the key cytokines reported to be

involved in neutrophil accumulation in the lungs during bacterial infections.38 Additionally, virulent pathogens such as _Francisella tularensis_ is known to inhibit IL-12 production in the

lung to promote infection.39, 40 Hence, we investigated if AB-8879 differentially regulates IL-12 production and thereby affect the neutrophil numbers. As shown in Supplementary Figure S5d,

both AB-8879 and AB-19606 elicited comparable levels of IL-12p70 in the BAL at both 4 and 24 h after infection. Collectively, these data suggest that the defect in the neutrophil

recruitment/numbers observed during infection with AB-8879 is unlikely due to the defect in the secretion of chemoattractants. NLRP3 INFLAMMASOME MACHINERY CONTRIBUTES TO THE HOST DEFENSE TO

CLINICAL ISOLATE, BUT IS DISPENSABLE FOR PROTECTION AGAINST THE TYPE STRAIN OF _A. BAUMANNII_ We next assessed the role of NLRP3 inflammasomes in the host defense to _A. baumannii_

infection. WT or Nlrp3−/− mice were intranasally infected with the clinical isolate AB-8879 or with type strain 19606 (5 × 107 CFU). The lungs were isolated at 24 h after infection and

bacterial load was determined. WT and Nlrp3−/− mice infected with AB-19606 strain had comparable bacterial load in the lungs (Figure 7a), possibly due to only minimal inflammasome activation

by the type strain (Figure 4a,b). However, Nlrp3−/− mice infected with AB-8879 had significantly higher bacterial burden in the lungs at 24 h after infection as compared with the

AB-8879-infected WT mice (2.2-fold difference, _P_=0.0001) (Figure 7a). This was also associated with a similar increase in bacterial load in the spleens of Nlrp3−/− mice infected with

AB-8879 (1.5-fold difference, _P_=0.002) (Figure 7b), indicating that NLRP3 may contribute to limit the extrapulmonary dissemination of AB-8879. Therefore, these results clearly demonstrate

that whereas NLRP3 pathway is not required for the host defense to AB-19606 infection, NLRP3 inflammasome pathway has an important role in the pulmonary host defense to AB-8879 infection.

NLRP3 INFLAMMASOME IS NEEDED FOR NEUTROPHIL RECRUITMENT TO THE LUNGS AT LATE STAGES OF INFECTION WITH _A. BAUMANNII_ CLINICAL ISOLATE IL-1β is a potent chemoattractant for neutrophils.41 We

hypothesized that higher bacterial burden in AB-8879-infected Nlrp3−/− mice (Figure 7) might be due to reduced IL-1β production (Figure 4d) and hence reduced neutrophil influx in the lungs

of these mice. Hence, we ascertained neutrophil numbers in the lungs/BAL from WT or Nlrp3−/− mice at 4 h (early time point) and 24 h (late time point) after infection. No difference in

neutrophils was observed between WT and Nlrp3−/− mice at 4 h after infection (Figure 8a,b), which was in accordance with the production of only very low levels of detectable IL-1β at this

time point. However, by 24 h after infection, AB-8879 induced IL-1β production in WT mice but not in Nlrp3−/− mice (Figure 4d). At this time point, 1.8-fold lesser neutrophils were present

in the lungs (_P_=0.01) and 1.3-fold lesser neutrophils were present in the BAL (_P_=0.007) of Nlrp3−/− mice as compared with WT mice (Figure 8a,b). Thus, increased susceptibility of

Nlrp3−/− mice to AB-8879 infection correlates with the presence of lesser neutrophil numbers in the lungs at 24 h after infection. These results suggest that NLRP3 contributes to the

neutrophil recruitment to the lungs during the late stages of infection (≥24 h after infection) leading to the control of _A. baumannii_ clinical isolate. ENHANCED NLRP3-DEPENDENT PRODUCTION

OF IL-1Β/IL-18 _IN VIVO_ IS MANIFESTED BY _A. BAUMANNII_ FROM DIFFERENT CLONAL GROUPS Having conclusively established that the clinical isolate AB-8879 (belonging to ICII) activates the

NLRP3 inflammasome _in vivo_, we next wanted to assess whether other virulent clinical isolates belonging to _A. baumannii_ ICI or ICII are also capable of doing so. For investigating

whether the enhanced inflammasome activation by AB-8879 _in vivo_ is a conserved phenomenon, we used another XDR-clinical isolate, _A. baumannii_ 40 (AB-40), belonging to IC clonal group

I,33 as a representative candidate. We found that the AB-40 was also capable of inducing enhanced IL-1β and IL-18 release in BAL of mice, the levels of which were comparable to those

produced by the AB-8879 (Figure 9a,b). Additionally, the production of IL-1β and IL-18 was abrogated in the lungs of AB-40-infected Nlrp3−/− mice. This result suggests that enhanced

inflammasome activation _in vivo_ may be a common feature of virulent clinical isolates of _A. baumannii_ belonging to both ICI and II. DISCUSSION Although the epidemiology and antibiotic

resistance traits of _A. baumannii_ clonal groups are well studied, the pathogenesis mechanisms and what determines the clinical success of ICI and II clones are not studied yet in detail.

Our study is the first report to demonstrate that clinically relevant _A. baumannii_ impairs pulmonary neutrophil recruitment, which may potentially contribute to their clinical success such

as higher pulmonary bacterial load and persistence. Our study has further demonstrated that clinical isolates of _A. baumannii_ differentially activates the host innate immune NLRP3

inflammasome pathway in comparison with type strain, which could potentially account for the uncontrolled inflammation and lung injury seen in _A. baumannii_ patients with compromised immune

system. Our study thus revealed a previously unappreciated role for NLRP3 inflammasome pathway in the host pulmonary defense to _A. baumannii_ clinical isolate. Early neutrophil recruitment

to the lungs was previously shown to be critical for _A. baumannii_ clearance and depletion of neutrophils was associated with higher bacterial load, extrapulmonary dissemination and severe

disease in the infected mice.11, 14, 15 Our study demonstrates a novel finding that the clinical isolate AB-8879 belonging to ICII evades this critical step controlling _A. baumannii_

pneumonia by reducing the early neutrophil recruitment to the lungs in comparison with the type strain. This differential interaction with the neutrophils could potentially account for the

higher lung bacterial load, persistence and extrapulmonary dissemination displayed by the clinical isolate belonging to ICII. Such delayed/reduced neutrophil recruitment to the lungs and

enhanced replication of bacteria has been previously reported for virulent bacterial pathogens such as _Yersinia pestis_.42 Yet another study has shown that A/J mice were more susceptible to

_A. baumannii_ pulmonary infection compared with B6 mice because A/J mice exhibited delayed early recruitment of neutrophils to the lungs that led to higher bacterial burden even at 72 h

after infection.13 Thus, apart from further highlighting the importance of early lung neutrophil recruitment in the control of _A. baumannii_ pneumonia, our study also suggests that

impairment of early neutrophil recruitment might be a virulence-related trait of _A. baumannii_ clinical isolates. Furthermore, we also show that at least one of the consequences of this

delayed neutrophil recruitment and higher bacterial load could be the differential ability of the clinical isolate to activate NLRP3-dependent IL-1β/IL-18 production. Indeed, a previous

report showed that higher BAL IL-1β correlated with higher bacterial burden in the lung alveoli of _A. baumannii_ pneumonia patients.21 The authors further went on to conclude that higher

BAL IL-1β levels could be a marker of ongoing inflammation because of persistent bacterial infection despite antibiotic therapy. Thus, our study could also mechanistically explain the

correlation of higher IL-1β levels with bacterial persistence and inflammation in patients having virulent clinically relevant _A. baumannii_ infections. We therefore propose that

inflammasome activation might be a second-line of host defense to clinically relevant virulent _A. baumannii_ that evades the initial neutrophil-mediated defense mechanisms, although further

studies are warranted to confirm this. On the other hand, it is also possible that enhanced bacterial burden alone cannot simply explain for the heightened inflammasome activation exhibited

by AB-8879, but rather the virulence attributes (such as bacterial-derived secreted virulence factors, toxins, etc.) or antibiotic resistance pattern of the clinical isolates could also

have a role in the enhanced activation of inflammasome pathway. To shed some light into this matter, we performed a comparative genomic analysis of AB-19606 with AB-8879 (Table 1 shows the

general features of each of the strains). We found that one of the major differences between the strains is the presence of multiple antibiotic resistance genes in AB-8879 in comparison with

AB-19606 (17 genes and 7 genes in AB-8879 and AB-19606, respectively). We further focused on putative genomic islands (Supplementary Figure S6a,b) and virulence genes (Table 2 and

Supplementary Figure S6c). Our analysis found that several virulence genes belonging to gene categories such as capsule, and heme utilization were unique to AB-8879 (Table 2). Supplementary

Figure S6c summarizes the number of virulence genes found only in AB-19606 or AB-8879 and also the number of genes that are common to both strains. The functional significance of these

unique genes in AB-8879 and whether or how they contribute to the differential activation of inflammasome pathway is a topic of future research. In this context, it is also worth noting that

although clinical isolate elicited enhanced activation of inflammasome pathway in comparison to type strain _in vivo_, both clinical and type strains activated inflammasome pathway to

comparable levels in BMDMs _ex vivo_. This result suggests that the differential interaction of the clinical isolate with the immune system may have a major role in the enhanced inflammasome

activation elicited by the clinical isolate _in vivo_. There is also a possibility that the clinical isolate secretes/produces virulence factors _in vivo_, but not _in vitro_, which

subsequently activates the inflammasome pathway differentially. The recruitment of neutrophils to the lung during infection is a multistep process coordinated by the production of

chemoattractants, and expression of adhesion molecules by endothelial cells near the site of inflammation/infection.43 As we found that there was no significant difference in the BAL levels

of chemoattractants such as GROα, MIP-2, CXCL5, and IL-12p70 among AB-8879 and AB-19606, we reason that AB-8879 impairs neutrophil recruitment targeting one or more steps other than the

inhibition of chemoattractant production. _A. baumannii_ infection induces lung inflammation and cell death, and this was proposed to account for the lung injury observed in experimental

murine models and patients with _A. baumannii_ pneumonia.44, 45 Higher levels of LDH observed in the lungs of AB-8879-infected mice in our study may indicate that the clinical isolate

induces more cell death/lung damage in comparison with the type strain. In addition, the cell death induced by AB-8879 was reduced in Nlrp3−/− mice implying that NLRP3 pathway contributes to

the cell death/lung damage during _A. baumannii_ infection. We also found that the IL-1β/IL-18 production during _A. baumannii_ infection occurs via cell death inducing caspase-1 and

caspase 11-dependent pathways. Caspase-11 is associated with the noncanonical inflammasome pathway that is involved in NLRP3-dependent activation of caspase-1, release of IL-1β/IL-1α, and

inflammatory cell death (pyroptosis) in response to various Gram-negative bacterial pathogens.32, 46, 47 It will be important to tease out the relative contributions of caspase-1 and

caspase-11 in the induction of pyroptosis during clinically relevant _A. baumannii_ infections. Our study revealed that AB-8879 caused enhanced activation of NLRP3 inflammasome pathway in

comparison with AB-19606. We also demonstrate that, besides contributing to cell death/lung damage, NLRP3 inflammasome is also required for the pulmonary host defense against the clinical

isolate AB-8879, but not AB-19606. Thus, inflammasome pathway may likely have a dual role (either protective or deleterious or both) in _A. baumannii_ infection depending on the immune

status of the host and also virulence of the bacteria. In fact, most _A. baumannii_ infections occur in patients with underlying disease conditions or compromised immune status, suggesting

that variability in the host immune response may contribute to the risk or severity of infection. Assessing the role of NLRP3 inflammasome in neutropenic mouse infection models (that

resembles the immunocompromised condition) will be helpful to clarify this point further. Our study showed that another clinical isolate belonging to ICI (AB-40) also activates

NLRP3-inflammasome-dependent IL-1β/IL-18 to the same level as AB-8879. Thus, our experiments provide reasonable evidence to suggest that heightened inflammasome activation is a common

feature of clinically relevant _A. baumannii_ isolates. Further studies using additional isolates belonging to ICI and II will be needed to determine the wider occurrence of

clone/strain/isolate-specific inflammasome response to _A. baumannii_ virulent infections. Important role of IL-1-associated pathways in pneumonia has been documented for various bacterial

infections.48 The effects of IL-1 are tightly controlled by two naturally occurring inhibitors, antagonist of IL-1 receptor and IL-1R type II (IL-1RII). Antagonist of IL-1 receptor is an

anti-inflammatory protein that counteracts the uncontrolled effects of proinflammatory responses to reduce the destructive immunopathology. Indeed, several studies have shown that

polymorphism of antagonist of IL-1 receptor gene is associated with respiratory injury/pneumonia in patients. Some examples includes: reduced levels of IL-1RA have been identified as a

marker for pneumonia in the elderly patients;49 genetic polymorphism of IL-1RA pathway is associated with pathophysiology of invasive pneumococcal disease in patients;50 Variable number

tandem repeat polymorphism within the intron 2 of _IL-1ra_ gene was associated with respiratory injury in children who had community-acquired pneumonia,51 etc. More importantly, a previous

study suggested that IL-1RA polymorphism is associated with patients with multidrug-resistant-_A. baumannii_-related pneumonia.22 Thus, the expression levels/polymorphism of IL-1-associated

pathway molecules could be considered as a marker for disease severity in _A. baumannii_ pneumonia patients. Collectively, our study demonstrates the importance of NLRP3 inflammasome pathway

in the immunity and pathogenesis of clinically relevant _A. baumannii_ infections. This study also opens the possibility that therapies targeted at modulating inflammasome pathway could be

considered as a viable option for either promoting bacterial control or for ameliorating the pathologic consequences associated with virulent clinically relevant _A. baumannii_ infections.

ETHICS STATEMENT All animal experiments were performed in strict accordance with the prevailing Singapore National Advisory Committee for Laboratory Animal Research (NACLAR) guidelines and

approved by Sing-Health Institutional Animal Care and Use Committee, Singapore (Protocol No.: 2013/SHS/863). NACLAR was established to develop national guidelines for the care and use of

animals for scientific purposes in Singapore. >MICE Eight–twelve-week-old female mice were used for all the mice infection experiments. C57BL/6J WT mice were purchased from the Biological

Resource Center (Agency for Science, Technology, and Research (A*STAR), Singapore). Nlrp3−/−,52 Asc−/−,53 Casp-1−/−,54 Casp-11−/−,54 and Casp-1/11−/−55 mice were used for the experiments.

All mutants were backcrossed to C57BL/6J background for at least 10 generations. REAGENTS AND CELL TREATMENT Tryptic soy broth (22092), tryptic soy agar (22091) and polymyxin B (P4932) were

purchased from Sigma-Aldrich (St. Louis, MO). LDH Cytotoxicity Kit (88953) was obtained from Pierce (Rockford, IL). LPS (EK-3701) was purchased from Invivogen (San Diego, CA) and was used at

100 ng ml−1 where indicated. ATP (A2383) was purchased from Sigma-Aldrich and was used at 5 mM where indicated. ELISA Kits for IL-1β (BMS6002TWO), IL-18 (BMS618/3), and tumor necrosis

factor-α (BMS607HS) were purchased from e-Bioscience (San Diego, CA). MIP-2 ELISA Kit (SMM200) was purchased from R&D (Minneapolis, MN). Mouse IL-1α ELISA Kit (ab113344) was obtained

from Abcam (Cambridge, MA). Caspase-1 antibody (AG-20B-0042-C100) was obtained from AdipoGen Life Sciences (San Diego, CA) and was used at 1:1,000 dilution for western blot. Neutrophil

Elastase Activity Kit was purchased from Cayman Chemical (Ann Arbor, MI) (600610). MPO ELISA Kit (ab155458) was purchased from Abcam. MPO antibody (Ab-9535) was purchased from Abcam and was

used at 1:25 dilution for immunohistochemistry (IHC) and immunocytochemistry (ICC). Wright–Giemsa stain (WG128) was purchased from Sigma-Aldrich. Lipofectamine 3000 (L3000015) was obtained

from Thermo Fisher Scientific (Waltham, MA) and was used as per the manufacturer’s instructions. Luciferase Kit (E1910) was obtained from Promega (Madison, WI). ATP ELISA Kit (ABIN627439)

was purchased through antibodies-online.com. All kits were used as per the manufacturer’s instructions. CA-074 Me (BML-P1126-0001) was purchased from Enzo Life Sciences (Farmingdale, NY).

APDC (sc-202408A) was purchased from Santa Cruz (Dallas, TX), KCl (P5405) and cytochalasin D (C8273) from Sigma-Aldrich. The reagents were used at following concentrations: CA-074 Me, 25 μM;

APDC, 100 μM; KCl, 50 mM; cytochalasin D, 5 μM. The cells were treated with these reagents for 1 h before infection and were maintained in these reagents throughout the course of the

experiments. BACTERIAL STRAINS AND GROWTH CONDITIONS AB-8879 and AB-40 isolates were cultured from skin tissue specimen and blood specimen of a burns patient, respectively.26, 27, 33 Both

AB-8879 and AB-40 were XDR, and susceptible to only polymyxin B or tigecycline. _A. baumannii_ ATCC 19606, AB-8879, and AB-40 strains were grown overnight in Tryptic soy broth supplemented

with 25 μg ml−1 streptomycin at 37 °C with shaking at 220 r.p.m. GENERATION OF BMDM BMDMs from indicated mice were isolated and cultured as described before.17 Briefly, bone marrow was

collected from the femur and tibia of 6–8-week-old mice. The bone marrow cells were cultured in Dulbecco's modified Eagle’s medium (Gibco, Waltham, MA) supplemented with 10% fetal calf

serum (Gibco), penicillin–streptomycin–amphoterecin (Thermo Fisher Scientific) and 30% L929 cell culture supernatants containing murine macrophage colony-stimulating factor. At day 7,

nonadherent cells were washed off and the adherent macrophages were removed using cell scrapers and used for further experiments. _IN VITRO A. BAUMANNII_ INFECTION BMDMs (0.5 × 106) from

indicated mice were seeded in a 24-well plate. Cells were pretreated with 100 ng ml−1 LPS overnight where mentioned. The cells were washed with phosphate-buffered saline (PBS) before

infection. Overnight grown cultures of _A. baumannii_ were harvested by centrifugation (3,000 _g_, 10 min), washed two times with PBS, and finally resuspended in Dulbecco’s modified Eagle’s

medium containing 10% fetal bovine serum medium. Cells were infected at MOI 100. To synchronize entry, the bacteria were centrifuged at 600 _g_ for 5 min and were incubated with cells for 2 

h. Subsequently, the cells were washed three times with PBS. Cells were then treated with polymyxin B (50 μg ml−1) for 1 h to kill the extracellular bacteria. Cells were washed three times

with PBS and either lysed in PBS containing 0.1% Triton X-100 and plated on trypticase soy agar (TSA) streptomycin plates to assess cell invasion (by calculating the CFU) or maintained in

medium with 5 μg ml−1 of polymyxin B for the rest of the duration of the experiment. _IN VIVO A. BAUMANNII_ INFECTION Mice were infected with _A. baumannii_ (AB-8879 or AB-19606)

intranasally as described earlier.15 Briefly, bacteria were grown overnight in tryptic soy broth supplemented with 25 μg ml−1 streptomycin. Next morning the cultures were reinoculated in

tryptic soy broth at 1%. After 3 h, the bacteria were washed and processed as described in the previous section. The bacteria were resuspended at indicated CFUs in 40 μl PBS. Actual inoculum

concentrations were determined by plating serial dilutions on TSA plates supplemented with 25 μg ml−1 streptomycin. Mice were anesthetized by administering ketamine/xylazine

intraperitoneally and were subsequently infected intranasally with _A.baumannii_. _IN VIVO_ BACTERIAL LOAD ENUMERATION For enumeration of bacterial CFU in various organs, mice were killed at

indicated time points after infections. Various organs were removed aseptically and were homogenized in 0.1% Triton X-100. Serial dilutions of the lysates were plated on TSA streptomycin

plates for CFU enumeration. For enumeration of bacterial CFU in blood, blood was collected from the submandibular vein in EDTA-coated tubes. Serial dilutions of blood were plated on TSA

streptomycin plates to enumerate bacteria. COLLECTION OF BAL For the aspiration of BAL, trachea was exposed through a midline incision. Lungs were lavaged 4–5 times with 1 ml PBS injected

through the trachea, as described previously.15 BAL fluid was spun at 2,000 r.p.m. for 10 min. The cells were processed for IHC, ICC and Giemsa staining as described below. The supernatant

was frozen at −80 °C and was analyzed for various cytokines as described below. GIEMSA STAINING, IHC AND ICC BAL was spun at 2,000 r.p.m. for 10 min. The cell pellet was resuspended in

PBS–bovine serum albumin 1% and was spun onto Superfrost Plus Slides (J1800AMN2; Thermo Fisher Scientific) using Cytospin 3 centrifuge. Slides were prepared for ICC or for Giemsa staining.

ICC staining for MPO analysis was carried out in the histopathology laboratory at The Advanced Molecular Pathology Laboratory (AMPL), Institute of Molecular and Cell Biology (IMCB),

Singapore. IHC staining of MPO in lungs was carried out as follows. Lungs were insufflated with 10% neutral-buffered formalin via trachea and removed _en bloc._ The lungs were kept in 10%

neutral buffer formalin until further processing for histological studies, which was carried out in the histopathology laboratory at AMPL, IMCB Singapore as described above. Lungs were

embedded in paraffin blocks. Serial sections (4–5 μm) were cut and processed for MPO staining by IHC. ICC and IHC slides were scanned at × 20 using Leica SCN400 Slide Scanner (Leica

Microsystems, Wetzlar, Germany). Images were exported to Slidepath Digital Image Hub (Leica Microsystems) for viewing. Images were analyzed using the Slidepath Tissue IA 2.0 Software (Leica

Microsystems). Data were collated using Microsoft Excel. Giemsa-stained slides were examined under light microscope at × 100 for differential cell counts. Hundred cells per slide were

counted and the percentage of neutrophils and macrophages was determined. CYTOKINE ANALYSIS For the _in vitro_ experiments, cell culture supernatants were collected at different time points

after infection. The cytokines were analyzed using ELISA Kits according to manufacturer’s instructions. For the analysis of cytokines produced during _in vivo_ infection, BAL was collected

from mice at various time points after infection, as already described. The lavage was spun at 2,000 r.p.m. for 10 min. The supernatant was concentrated using Vivaspin 500 columns (5 kDa

cutoff, VS0012; Sartorius, Gottingen, Germany). The supernatant was analyzed for various cytokines using ProcartaPlex Multiplex Immunoassay from eBioscience by the Immunomonitoring Platform,

SIgN, A*STAR, Singapore. For the analysis of MPO by ELISA, lungs from mice were obtained at indicated time points and homogenized in 0.1% Triton X-100. The lysates were then spun at 2,000 

r.p.m. for 10 min. The supernatants were frozen at −80 °C and were later analyzed for MPO by ELISA as per the manufacturer’s instructions. RNA ISOLATION AND QUANTITATIVE REVERSE

TRANSCRIPTION-PCR At designated time points, BMDMs were lysed in RLT buffer and RNA was isolated using RNeasy Kit (74106; Qiagen). cDNA synthesis was carried out by iScript cDNA Synthesis

Kit (170-8891; Bio-Rad, Hercules, CA) as per the manufacturer’s instructions. Primer sequences for various genes amplified were as described in Bouwman _et al._56 Reactions were performed in

Roche Light Cycler 480 Real-time PCR system under the following conditions: 94 °C/2 min for 1 cycle; 94 °C/30 s, 50 °C/30 s, 72 °C/60 s for 40 cycles; 72 °C/7 min for 1 cycle. Results were

analyzed using delta-delta−Ct method. WESTERN BLOT BMDMs (2 × 106) were seeded in a 24-well plate and infected with _A.baumannii_ as described above. At 24 h after infection, cell culture

supernatants were collected and concentrated using Vivaspin 500 Columns (cutoff 5 kDa, VS0012; Sartorius). The concentrated supernatants were separated by 12% denaturing sodium dodecyl

sulfate-polyacrylamide gel electrophoresis. Western blotting was performed on the separated proteins transferred to PVDF membrane using standard procedures. Blots were probed for caspase-1

p20. The signal was developed using an ECL Western Kit (GE Healthcare, Chicago, IL). FACS STAINING Mice were infected with AB-8879 or AB-19606. At 4 h after infection, mice were killed and

BAL was collected on ice. BAL was spun at 1,500 r.p.m. for 10 min at 4 °C. The cell pellet was resuspended in PBS- bovine serum albumin 1% and stained with the following antibodies: PE CD11b

(130-098-087; Miltenyi Biotec, Bergisch Gladbach, Germany) and FITC Ly6G (130-102-934; Miltenyi Biotec) as per the manufacturer’s instructions. The cells were subsequently fixed in 4%

paraformaldehyde. The samples were run on Fortessa FACS analyzer (BD Biosciences, NJ) and the analysis was carried out with the FlowJo Software (FlOWJO, LLC, Ashland, OR). GENOMIC ANALYSIS

Genome sequence of AB-19606 was obtained from GenBank (accession number JMRY00000000.1). Genome sequence of AB-8879 is available in our local database. Genome annotation was performed using

PROKKA on the assembled contigs of AB-8879. The annotated genomes of AB-8879 and AB-19606 in GenBank format were used in IslandViewer3 to predict genomic islands using _A. baumannii_

multidrug-resistant-ZJ06 complete genome as reference. SRST2 is used to identify the antibiotic resistance genes found in AB-8879 and AB-19606 using ARGannot resistance gene database

included in the program. SRST2 was used to identify virulence genes in _A. baumannii_ AB-8879 and AB-19606. To generate the virulence genes database for SRST2, DNA sequences of full data set

was downloaded from the virulence factor database. Then, the virulence genes by _A. baumannii_ were extracted and clustered at 90% nucleotide identity. SRST2 uses sequencing reads for genes

typing. Paired-end reads from Illumina sequencing for AB-8879 is available in our database. Single-ended 100 bp reads from Illumina HiSeq 2000 for AB-19606 was extracted using SRA Toolkit

under the accession number SRR2180354 from NCBI. STATISTICAL ANALYSIS _P_ values were determined by unpaired two-tailed Student’s _t_-test. Analysis of variance test was used in multiple

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activation by _Campylobacter jejuni_. _J. Immunol._ 193, 4548–4557 (2014). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank the team of Mutant Mouse Collection

Core Service (Singapore Immunology Network, SIgN) for assistance in breeding and husbandry of some mutant mice and Immunomonitoring Platform (SIgN) for luminex analysis. We also thank Dr Ong

Twee Hee (National University of Singapore, Singapore) for helping with the genome analysis of the strains. This work was supported by Duke-NUS Signature Research Program funded by Agency

for Science Technology and Research (A*STAR) & Ministry of Health, Singapore (to B.S.); National Medical Research Council Centre Grant (NMRC/CG/016/2013) (to A.L.K.), and Singapore

Immunology Network (SIgN) core funds (to A.M.). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Program in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore N

Dikshit, S D Kale, V Balamuralidhar, P Kumar, A L Kwa & B Sukumaran * Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore H J

Khameneh & A Mortellaro * Life Sciences Institute, National University of Singapore, Singapore, Singapore C Y Tang * Department of Pharmacy, Singapore General Hospital, Singapore, T P

Lim & A L Kwa * Sing Health Duke-NUS Medicine Academic Clinical Programme (MED ACP), Singapore, Singapore T P Lim & A L Kwa * Department of Infectious Diseases, Singapore General

Hospital, Singapore, Singapore T T Tan * Department of Pharmacy, National University of Singapore, Singapore, Singapore A L Kwa Authors * N Dikshit View author publications You can also

search for this author inPubMed Google Scholar * S D Kale View author publications You can also search for this author inPubMed Google Scholar * H J Khameneh View author publications You can

also search for this author inPubMed Google Scholar * V Balamuralidhar View author publications You can also search for this author inPubMed Google Scholar * C Y Tang View author

publications You can also search for this author inPubMed Google Scholar * P Kumar View author publications You can also search for this author inPubMed Google Scholar * T P Lim View author

publications You can also search for this author inPubMed Google Scholar * T T Tan View author publications You can also search for this author inPubMed Google Scholar * A L Kwa View author

publications You can also search for this author inPubMed Google Scholar * A Mortellaro View author publications You can also search for this author inPubMed Google Scholar * B Sukumaran

View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to B Sukumaran. ETHICS DECLARATIONS COMPETING INTERESTS The authors

declared no conflict of interest. ADDITIONAL INFORMATION AUTHOR CONTRIBUTIONS Conceived and designed the experiments: N.D. and B.S. Performed the experiments: N.D., S.D.K., H.J.K., V.B.,

C.Y.T., P.K. and L.T.P. Analyzed the data: N.D., T.T.T., A.L.K., A.M. and B.S. Contributed reagents/materials: A.M. and A.L.K. Wrote the manuscript: N.D. and B.S. Critically revised the

manuscript: A.M. and H.J.K. SUPPLEMENTARY MATERIAL is linked to the online version of the paper SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE LEGENDS (DOC 31 KB) SUPPLEMENTARY FIGURES (PDF

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Kale, S., Khameneh, H. _et al._ NLRP3 inflammasome pathway has a critical role in the host immunity against clinically relevant _Acinetobacter baumannii_ pulmonary infection. _Mucosal

Immunol_ 11, 257–272 (2018). https://doi.org/10.1038/mi.2017.50 Download citation * Received: 22 January 2017 * Accepted: 15 April 2017 * Published: 14 June 2017 * Issue Date: January 2018 *

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