Figures
Abstract
Burkholderia pseudomallei causes the tropical infection melioidosis. Pneumonia is a common manifestation of melioidosis and is associated with high mortality. Understanding the key elements of host defense is essential to developing new therapeutics for melioidosis. As a flagellated bacterium encoding type III secretion systems, B. pseudomallei may trigger numerous host pathogen recognition receptors. TLR5 is a flagellin sensor located on the plasma membrane. NLRC4, along with NAIP proteins, assembles a canonical caspase-1-dependent inflammasome in the cytoplasm that responds to flagellin (in mice) and type III secretion system components (in mice and humans). In a murine model of respiratory melioidosis, Tlr5 and Nlrc4 each contributed to survival. Mice deficient in both Tlr5 and Nlrc4 were not more susceptible than single knockout animals. Deficiency of Casp1/Casp11 resulted in impaired bacterial control in the lung and spleen; in the lung much of this effect was attributable to Nlrc4, despite relative preservation of pulmonary IL-1β production in Nlrc4−/− mice. Histologically, deficiency of Casp1/Casp11 imparted more severe pulmonary inflammation than deficiency of Nlrc4. The human NLRC4 region polymorphism rs6757121 was associated with survival in melioidosis patients with pulmonary involvement. Co-inheritance of rs6757121 and a functional TLR5 polymorphism had an additive effect on survival. Our results show that NLRC4 and TLR5, key components of two flagellin sensing pathways, each contribute to host defense in respiratory melioidosis.
Author Summary
Melioidosis is an infection caused by Burkholderia pseudomallei, a bacterium that is found in tropical soil and water. Melioidosis can present in a variety of ways, but lung involvement is common and usually severe. The host response to infection governs outcome. In this study, we examined the role of two host sensors of bacterial components–TLR5 and NLRC4–to determine their necessity in respiratory melioidosis. Although both proteins are involved in detection of bacterial flagellin, in mice we defined specific and individual roles for TLR5 and NLRC4 in protecting against death from melioidosis. In humans with melioidosis involving the lung, genetic variation in these receptors also had independent associations with survival. These results underscore the importance of these elements of host defense in respiratory melioidosis and support further studies of the underlying mechanisms.
Citation: West TE, Myers ND, Chantratita N, Chierakul W, Limmathurotsakul D, Wuthiekanun V, et al. (2014) NLRC4 and TLR5 Each Contribute to Host Defense in Respiratory Melioidosis. PLoS Negl Trop Dis 8(9): e3178. https://doi.org/10.1371/journal.pntd.0003178
Editor: Pamela L. C. Small, University of Tennessee, United States of America
Received: February 24, 2014; Accepted: August 9, 2014; Published: September 18, 2014
Copyright: © 2014 West et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding was provided by National Institutes of Health (K08HL094759, U54AI057141, R01HL113382); the Puget Sound Partners for Global Health; the Parker B. Francis Foundation; and the Wellcome Trust. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Burkholderia pseudomallei is a tropical soil saprophyte and Tier 1 select agent that causes the infection melioidosis [1]. The bacterium may be inoculated through the skin, inhaled, or ingested. Although infection can manifest in myriad ways, pneumonia is identified in 50% of cases. Mortality from melioidosis ranges from 14–40% despite appropriate antibiotic treatment, and the risk of death is higher with pulmonary involvement [2], [3]. This indicates an urgent need for a better understanding of host-pathogen interactions in melioidosis and adjunctive immuno-modulatory therapies.
Innate immune mechanisms of recognition of invading bacteria include membrane-bound Toll-like receptors (TLRs) and cytosolic NOD-like receptors (NLRs) [4], [5]. These pathogen recognition receptors bind conserved pathogen associated molecular patterns and drive the host response. For example, as a Gram-negative, flagellated bacterium, B. pseudomallei is predicted to activate sensors of LPS (such as TLR4) and flagellin (such as TLR5). We have found that B. pseudomallei LPS is a TLR4 ligand that drives much of the innate immune response to B. pseudomallei, and that human genetic variation in TLR4 is associated with susceptibility to melioidosis [6]–[8]. We have also shown that B. pseudomallei activates TLR5, and that polymorphisms in TLR5 are associated with survival from melioidosis [9], [10], however TLR5-deficient mice have not been infected with B. pseudomallei to demonstrate the role of TLR5 in an experimental setting. These findings point to an important role for flagellin in activation of immune responses in melioidosis.
Whereas TLR5 detects flagellin at the cell surface, cytosolic flagellin is detected through NLRC4, an inflammasome that activates caspase-1 [11]. NLRC4 is one of a number of NLRs that can assemble a canonical caspase-1-dependent inflammasome that in turn cleaves pro-IL-1β and pro-IL-18 to their active forms and induces pyroptosis [5], [12]. More recent work identified murine NAIP5 and NAIP6 as direct flagellin sensors that signal through NLRC4 [13], [14]. NLRC4 also contributes to the sensing of bacterial components other than flagellin: murine NLRC4-NAIP1 and NLRC4-NAIP2 inflammasomes recognize bacterial type three secretion system (T3SS) needle and rod proteins, respectively [13]–[16]. In contrast to mice, humans have only a single NAIP, and in human U937 monocytes the NLRC4-NAIP inflammasome recognizes a T3SS needle protein but not flagellin [14]. The functional interpretation of the three NLRC4 agonists is similar – flagellin, rod, and needle protein are all believed to be accidentally injected into the cytosol by bacterial T3SS. This is in contrast to TLR5 detecting extracellular flagellin, the presence of which will not be strictly linked to a particular virulence trait. In addition to the canonical caspase-1-dependent inflammasome, a noncanonical inflammasome involving another inflammatory caspase, caspase-11, has recently been described in mice [17]. Caspase-11 protects mice from B. pseudomallei infection [18].
In this study, our primary objective was to determine the relative importance of NLRC4 in murine respiratory melioidosis in comparison to TLR5, and with respect to canonical and noncanonical inflammasomes. Our secondary objective was to test whether genetic variation in NLRC4 was associated with outcome in human respiratory melioidosis.
Methods
Ethics statement
All animal experiments were approved by the University of Washington Institutional Animal Care and Use Committee (protocol number 2982-03). The University of Washington complies with all applicable provisions of the federal Animal Welfare Act and with the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals. The University of Washington Human Subjects Division Institutional Review Board; the Ethical Review Committee for Research in Human Subjects, Ministry of Public Health, Thailand; and the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand approved the human genetic studies on subjects who had provided or whose next of kin had provided written informed consent for enrollment into clinical studies of melioidosis at the time of recruitment.
Bacteria
B. pseudomallei 1026b was grown in LB broth shaking in air at 37°C, washed twice, resuspended in PBS containing 20% glycerol, and frozen at −80°C. Immediately before each aerosol infection experiment, the freezer stock was thawed and diluted in PBS to the desired concentration, as previously described [19].
Mouse model of melioidosis
Animals.
Specific pathogen-free C57BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Tlr5−/− mice are previously described [20]. Nlrc4−/− mice were obtained from Vishva Dixit [21]. Casp1−/−Casp11−/− mice were obtained from Richard Flavell [22]. All mice were backcrossed at least six generations onto a C57BL/6 background and Tlr5−/−Nlrc4−/− mice were subsequently derived. Mice were housed in isolator cages with ad lib access to chow and water, and were monitored one to two times daily.
Infection.
Mice were exposed to aerosolized bacteria in a 24 port cylindrical nose-only exposure chamber (In-Tox Products, Moriarty, NM) [19]. Aerosols were generated by a MiniHeart Hi-Flo nebulizer (Westmed, Tucson, AZ) driven at 8 L/min with 7 L/min simultaneous dilution air for 10 minutes followed by 5 minutes washout period. Pressure and airflow were controlled by an AeroMP aerosol management platform (Biaera Technologies, Frederick, MD). Bacterial deposition in each experiment was determined from quantitative culture of lung tissue from four mice sacrificed immediately after infection. Animals were examined daily for illness or death and their clinical condition recorded. When indicated, abdominal surface temperatures were measured using a Ranger MX4P digital infrared thermometer (Raytek, Santa Cruz, CA, USA). Ill animals with temperatures <21.5°C or a combination of ruffled fur, eye crusting, hunched posture and lack of resistance to handling were deemed terminal and euthanized. Spontaneous death was not required as an endpoint.
Bacterial quantification.
Twenty four or forty eight hours after infection mice were sacrificed. The left lung and spleen each were homogenized in 1 mL sterile PBS. One hundred microliters each of homogenate and 10-fold serial dilutions were plated in duplicate on LB agar or Ashdown's medium. Colonies were counted after 2–4 days of incubation at 37°C or up to a week incubating at room temperature.
Lung histology and quantitative morphometry.
The right lung was fixed in 4% paraformaldehyde as previously described [19]. Lung tissue was embedded in paraffin, sectioned to expose maximum surface area, and stained with hematoxylin and eosin; sections were examined by a veterinary pathologist who was blinded to group assignment. The number of focal inflammatory lesions, area of each lesion, and total lung tissue area in one representative section from each mouse were determined using Nikon NIS-Elements software.
Cytokine measurements.
Left lung homogenates in PBS were diluted 1∶1 in lysis buffer containing 2× protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), incubated on ice for 30 min, and then centrifuged at 1500× g. Supernatants were collected and stored at −80°C until assayed for cytokines. Whole blood was centrifuged, serum removed and stored at −80°C until assayed. IFN-γ, IL-10, IL-12p70, IL-1β, IL-6, KC, and TNF-α were measured in lung homogenates and serum using a multiplex bead assay (Luminex, Austin, TX) and reagents purchased from R&D Systems.
Human subjects
Clinical cohort.
Human genetic analyses were performed on patients with culture-proven melioidosis admitted to Sappasithiprasong Hospital, Ubon Ratchathani, Thailand from 1999 through 2005 [8]–[10]. A study team screening patients with clinical signs of infection cultured sputum, blood, urine and other relevant samples (for example, abscess aspirates) for B. pseudomallei. Samples from some patients were independently submitted for culture by hospital clinicians. Pulmonary involvement was defined by a positive B. pseudomallei culture from the respiratory tract or radiographic evidence of a pulmonary infiltrate and a positive B. pseudomallei culture from another clinical specimen. Deaths were defined as those individuals who died during their hospitalization or were discharged home in extremis for palliative care. DNA was extracted from blood using a Nucleon BACC3 kit (GE Healthcare, Buckinghamshire, UK).
Polymorphism selection and genotyping
NLRC4 SNP identification and selection was performed using the Genome Variation Server (http://gvs.gs.washington.edu/GVS/). Coding SNPs in the gene and haplotype-tagging SNPs were selected. Within the region encompassed by 50,000 bases upstream and downstream of NLRC4, SNPs with a minor allele frequency ≥2% in populations identified as Japanese, Chinese and Asian were binned into groups with R2≥0.8 to identify haplotype-tagging SNPs. Genotyping was performed using an allele-specific primer extension method (Sequenom Inc., San Diego, CA, USA) with reads by a MALDI- TOF mass spectrometer [8].
Statistics
Comparisons of two and three groups of data expected to follow a normal distribution were made using Student's t test and ANOVA with a Bonferroni post-test, respectively. CFUs were log10 transformed before analysis. Survival analyses were performed with the log rank test. SNPs were tested for deviation from Hardy-Weinberg equilibrium using the exact test. The association of genotype with death was performed using a Chi square test or, for contingency tables with cell counts <10, the exact test. For multivariate analysis of genetic associations, logistic regression was performed adjusting for age, gender, diabetes, renal disease, or liver disease. A conservative Bonferroni correction was not performed as the variants are unlikely to be independent. Effect modification was assessed by testing the incorporation of an interaction variable into the regression model, using the likelihood ratio test. Statistics were performed with GraphPad Prism 5.0f (San Diego, CA) or Stata 11.2 (College Station, TX). A two sided p value of ≤0.05 was considered significant.
Results
Given our previous identification of a strong association between a nonsense TLR5 polymorphism that renders TLR5 insensitive to flagellin and survival from melioidosis [9], we examined whether the presence of Tlr5 in murine melioidosis altered survival. We infected mice with 361 CFU B. pseudomallei per lung, a dose that approximates the median lethal dose (Figure 1A). We found that Tlr5−/− mice had significantly poorer survival than wild type mice, a phenotype that contrasts with that observed in Tlr2−/− or Tlr4−/− mice [23].
Wild type (WT), Tlr5−/−, Tlr5−/−Nlrc4−/−, or Nlrc4−/− mice were infected with A) 361 CFU/lung B) 400 CFU/lung or C) 91 CFU/lung aerosolized B. pseudomallei 1026b and monitored at least daily for survival. N = 5–7 per group (A), 4 per group (B), and 5 per group (C). *, p≤0.05 and **, p≤0.01 for Tlr5−/− compared to wild type mice;). ††, p≤0.01 for Tlr5−/−Nlrc4−/− compared to wild type mice; §§, p≤0.01 for Nlrc4−/− compared to wild type mice. No significant differences in survival were noted for Tlr5−/− or Nlrc4−/− compared to Tlr5−/−Nlrc4−/− mice.
We next asked how the absence of Nlrc4 modulated this phenotype. We infected Tlr5−/− and Tlr5−/−Nlrc4−/− mice with a similar dose (400 CFU/lung) of B. pseudomallei but found no difference in survival between mouse strains (Figure 1B). This finding suggested that the absence of flagellin sensing at the cell surface sufficiently impaired the host response such that impaired cytosolic detection of the pathogen did not substantially impact survival further. We then tested whether lack of Nlrc4 alone altered survival in respiratory melioidosis, and how this differed from combined deficiency of Tlr5 and Nlrc4. To increase the sensitivity of our model, we chose a lower inoculum that is non-lethal to wild type mice (91 CFU/lung). We found that Nlrc4-deficient mice were more susceptible to melioidosis than wild type mice, consistent with results from Ceballos-Olvera [24], but that there was no difference in survival between Nlrc4−/− mice and Tlr5−/−Nlrc4−/− mice (Figure 1C). Together, these experiments demonstrate that TLR5 and NLRC4 each contribute to host defense in murine respiratory melioidosis.
Caspase-11 has recently been identified as a component of the noncanonical, caspase-1-independent inflammasome. We and others have found that Casp1−/−Casp11−/− mice infected with B. pseudomallei by the respiratory route failed to control infection (unpublished data, [24], [25]). To examine the effects of NLRC4 relative to other caspase-1- and caspase-11- dependent inflammasomes, we directly compared bacterial burdens in organs of wild type, Nlrc4−/−, or Casp1−/−Casp11−/− mice infected with B. pseudomallei. Twenty four hours after an inoculum of 314 CFU/lung, bacterial growth in the lungs of both Nlrc4−/− and Casp1−/−Casp11−/− mice was about 0.87 log10 CFU greater than in wild type mice, and there was no significant difference between CFU in Nlrc4−/− compared to Casp1−/−Casp11−/− mice (Figure 2). Forty eight hours after infection, bacterial growth in the lungs of both Nlrc4−/− and Casp1−/−Casp11−/− mice had increased significantly compared to wild type mice (by 1.87 log10 CFU and 2.69 log10 CFU, respectively). Despite a trend towards greater pulmonary bacterial burdens in Casp1−/−Casp11−/− mice than in Nlrc4−/− mice, this did not reach statistical significance. Bacterial burdens in the spleens are an indication of dissemination beyond the pulmonary compartment. Although bacteria were detectable 24 hours after infection, there were no significant differences between the three mouse strains. Forty eight hours after infection, CFU were significantly greater in Casp1−/−Casp11−/− mice compared to Nlrc4−/− mice which in turn had greater bacterial burdens compared to wild type mice. These data confirm that while deficiency of both caspase-1 and caspase-11 severely impairs control of B. pseudomallei replication in the lung, NLRC4 accounts for much of the inflammasome-dependent phenotype [24].
Wild type (WT), Nlrc4−/−, and Casp1−/−Casp11−/− mice were infected with 314 CFU/lung aerosolized B. pseudomallei 1026b. Lungs (A) and spleens (B) were harvested and quantitatively cultured 24 and 48 hours after infection. Data displayed are means ± SD and represent one of two comparable experiments. N = 4 per group per timepoint except N = 3 for Casp1−/−Casp11−/− mice at 24 h. *, p≤0.05, **, p≤0.01, and ***, p≤0.001 for Nlrc4−/− compared to wild type mice; †††, p≤0.001 for Casp1−/−Casp11−/− compared to wild type mice; §§§, p≤0.001 for Nlrc4−/− compared to Casp1−/−Casp11−/− mice.
We next evaluated selected cytokine and chemokine responses in the lungs of these mice (Figure 3). There were no differences in TNF-α or MIP-2 levels between mouse strains at 24 hours. As expected, IL-1β was very low in Casp1−/−Casp11−/− mice but was not impaired in Nlrc4−/− mice. Chemokine KC was higher in Nlrc4−/− mice compared to wild type and to Casp1−/−Casp11−/− mice. By 48 hours after infection, TNF-α levels in Casp1−/−Casp11−/− mice were significantly greater than wild type. MIP-2 and KC levels in Casp1−/−Casp11−/− and Nlrc4−/− mice were higher than in wild type mice. IL-1β was elevated in all mice compared to 24 hour levels, but was significantly elevated in Nlrc4−/− mice in comparison to wild type and to Casp1−/−Casp11−/− mice. In serum 24 hours after infection, TNF-α, MIP-2, and Il-1β levels were low but KC was readily detectable and higher in Nlrc4−/− mice compared to Casp1−/−Casp11−/− mice. At 48 hours, despite higher bacterial burdens in the spleens of Nlrc4−/− and Casp1−/−Casp11−/− mice compared to wild type mice, serum TNF-α and IL-1β remained uniformly low. In contrast, MIP-2 and KC levels increased substantially in both Nlrc4−/− and Casp1−/−Casp11−/− mice. In line with previously published data [24], these results point to non-NLRC4-mediated pathways of IL-1β production in the lung, but suggest that systemically, NLRC4 mediates TNF-α and IL-1β but not MIP-2 or KC release.
Wild type (WT), Nlrc4−/−, and Casp1−/−Casp11−/− mice were infected with 314 CFU/lung aerosolized B. pseudomallei 1026b. TNF-α, MIP-2, KC, and IL-1β were measured in lung homogenate (A–D) and serum (E–H) 24 and 48 hours after infection. Data displayed are means ± SD and represent one of two comparable experiments. N = 4 per group per timepoint except N = 3 for Casp1−/−Casp11−/− mice at 24 h. *, p≤0.05. **, p≤0.01, ***, p≤0.001.
Inhalation of B. pseudomallei results in scattered, dense cellular pulmonary infiltrates [19]. Histopathologic examination of the lungs of Nlrc4−/− and Casp1−/−Casp11−/− mice 24 hours after airborne infection with B. pseudomallei showed relatively similar sized neutrophilic infiltrates and percent of lung involved in these mice compared to wild type mice although there was minor variation in morphologic features, such as earlier evidence of nuclear fragmentation in Casp1−/−Casp11−/− mice (Figure 4). However, at 48 hours, inflammation was more severe, particularly in Casp1−/−Casp11−/− mice, which displayed larger and necrotic parenchymal lesions that lacked identifiable intact inflammatory cells.
Wild type (WT), Nlrc4−/−, and Casp1−/−Casp11−/− mice were infected with 314 CFU/lung or 194 CFU/lung aerosolized B. pseudomallei 1026b in two separate experiments. Lungs were fixed and stained with hematoxylin and eosin 24 and 48 hours after infection. The average size of each focal pulmonary infiltrate normalized to measures from wild type mice at 24 hours (A) and the fraction of total lung area that was abnormal (B) were quantified for each group of mice. Data displayed are means ± SD from the two experiments. N = 4–7 per group per timepoint. *, p≤0.05, **, p≤0.01. (C) Representative histologic features of pulmonary infection with B. pseudomallei. At 24 h post-infection lesions are similar between the groups in pattern, distribution and size but have slightly varying morphologic features. Inflammatory foci in WT mice are composed of moderately dense aggregates of nearly pure, intact neutrophils that fill vessels and alveolar spaces but preserve architectural elements. In contrast focal inflammatory cell aggregates in Nlrc4−/− and Casp1−/−Casp11−/− while also predominately neutrophilic (with few mononuclear cells) are much more densely packed, slightly obscuring some normal structures and, in the case of Casp1−/−Casp11−/− showing early evidence of nuclear fragmentation. By 48 hours post-infection phenotypic differences are more distinct. Focal lesions within WT mice while modestly larger remain predominately neutrophilic with limited evidence of parenchymal tissue necrosis and relatively mild neutrophil condensation and fragmentation. Nuclear condensation and fragmentation is a hallmark of lesions in Nlrc4−/− mice at 48 h but parenchymal injury remains relatively modest with many alveolar wall outlines preserved and small airway involvement very limited. Mixed inflammatory cells surround these lesions. This contrasts with the severe injury evident in Casp1−/−Casp11−/− mice at 48 h. In these mice lesions are substantially larger and efface all or most parenchymal and vascular structures which are focally replaced by necrotic cells and a dark pink coagulum containing variably fine nuclear debris. This debris commonly extends into small airways. Intact inflammatory cells of any type are rarely evident within these lesions although adjacent vessels are surrounded by small numbers of intact mononuclear cells.
We have found that a human genetic polymorphism in TLR5 is associated with outcome from melioidosis [9]. Given the clear role for Nlrc4 in murine respiratory melioidosis, we investigated whether human genetic variation in the NLRC4 region was associated with death in human respiratory melioidosis. We genotyped five NLRC4 region single nucleotide polymorphisms (SNPs) (rs455060, rs212703, rs410469, rs462878, and rs6757121) selected as described in the methods in 173 melioidosis patients with clinical evidence of pulmonary involvement. The call rate for four SNPs was above 97.5%; one (rs212703) was discarded due to a low call rate. Fifty eight of the 173 subjects (34%) died. In survivors, no variant deviated from Hardy-Weinberg equilibrium. rs6757121 was associated with protection against death in a general genetic model, p = 0.012 (Table 1). Adjusting for age, sex, and pre-existing conditions, the effect was strongest in a dominant model [odds ratio (OR) 0.35, 95% CI:0.13–0.91, p = 0.03]. rs6757121 is located about 0.3 kb downstream of NLRC4 and occurs with a minor allele frequency of 10%.
We next tested whether our previously reported association between TLR51174C>T – a nonsense polymorphism that truncates the receptor in the extracellular domain rendering it non-responsive to flagellin – and survival in melioidosis [9] is also seen in the subset of melioidosis patients with respiratory disease. We found that the adjusted OR of death was 0.14, 95% CI 0.03–0.64, p = 0.01. To determine whether co-inheritance of this TLR5 variant and the NLRC4 region variant rs6757121 alters the risk of death from respiratory melioidosis, we assessed the effect of including both together in the model. The OR of death for each variant remained unchanged, although the effect of a cross-product interaction term could not be determined due to 100% survival in carriers of both variants. The estimated OR of death for carriers of both variants was 0.04, 95% CI: 0.006–0.27, p = 0.001 (Table 2). Together, these data show that the NLRC4 and TLR5 variants are each associated with survival and that co-inheritance of the variants has an additive but not synergistic effect.
Discussion
The results of our investigations show that NLRC4 and TLR5, key components of two flagellin sensing pathways, each contributes to host defense in murine respiratory melioidosis. We did not detect any additional impact of deficiency of both Nlrc4 and Tlr5 on survival. Furthermore, NLRC4 is responsible for much of the failure of pulmonary bacterial containment seen in caspase-1/-11-deficient mice. In humans, we show that an NLRC4 genetic variant is associated with survival in respiratory melioidosis, and there is an additive effect of co-inheritance of risk variants in TLR5 and NLRC4.
Recent investigations have demonstrated that NLRC4 is involved in recognition of several bacterial ligands such as components of the T3SS or flagellin, and this specificity is determined by various NAIPs [13]–[16]. In contrast, the only reported ligand of TLR5 is flagellin [4]. B. pseudomallei activates TLR5 [9] and aflagellated B. pseudomallei induces impaired TLR5-dependent NF-κB activation in vitro [unpublished data]. Our present results show that Tlr5−/− mice are more susceptible to B. pseudomallei in a model of respiratory infection, in contrast to deficiency in Tlr2, which actually confers resistance, or Tlr4, which has no apparent effect on survival [23]. Although MyD88 is an adapter molecule for all three of these TLRs, mice deficient in Myd88 show a similar phenotype to deficiency in Tlr5 after respiratory infection with B. pseudomallei [26]. Flagellin-sensing appears to be a crucial element of host defense in murine respiratory melioidosis. However, we have not observed significant impairment in TNF-α production from Tlr5−/− alveolar macrophages stimulated ex vivo with killed B. pseudomallei [unpublished data] and it is notable that our studies of murine respiratory infection with B. thailandensis (a related and flagellated but less virulent organism) have not shown any Tlr5-dependent phenotype [20]. Thus, in vitro data, and infections with model organisms may not fully recapitulate the complexity of in vivo infections with fully virulent B. pseudomallei.
Like TLR5, NLRC4 appears to play a central role in host defense in respiratory murine melioidosis. Interestingly, while NLRC4 detects flagellin from many bacterial species, it appears to not detect B. thailandensis (and presumably B. pseudomallei) flagellin [14], thus, the effect of NLRC4 in vivo may be attributable to T3SS sensing. B. pseudomallei expresses several T3SSs [27] and T3SS3 facilitates virulence in a number of ways [28]–[31]. The B. pseudomallei T3SS rod and needle proteins BsaK and BsaL, respectively, are detected in an NLRC4-dependent fashion in mice [15], [32]. Recent work by Bast et al demonstrates the importance of BsaK for NLRC4-dependent caspase-1 activation in B. pseudomallei-infected macrophages and for virulence in murine melioidosis [33]. Intriguingly, despite the different sensing functions of TLR5 and NLRC4, absence of only one sensor imparts significant clinical impairment; there is no additive effect on survival of combined Tlr5 and Nlrc4 deficiency in murine melioidosis, even at doses that are non-lethal to wild type mice.
NLRC4 is just one of many pathogen recognition receptors that activate the caspase-1-dependent inflammasome. The inflammasome processes pro-IL-1β and pro-IL-18 to their active forms and also induces pyroptosis, a caspase-1-dependent lytic cell death pathway. In our studies, Nlrc4−/− mice did not show a significant difference compared to Casp1−/−Casp11−/− mice with respect to bacterial replication in the lung following respiratory infection, but did show a difference in disseminated infection to the spleen, consistent with the work of Ceballos-Olvera et al [24]. This difference in dissemination may be due to caspase-11, which also has been implicated in defense against B. pseudomallei [18]. Relative to Casp1−/−Casp11−/− mice, Nlrc4−/− mice showed preserved pulmonary IL-1β production. These data raise the possibility that much of the early effect of inflammasome-dependent control of bacterial replication in the lung is primarily NLRC4-dependent and the function of NLRC4 may be due to pyroptosis or as-yet-undefined roles of NLRC4 rather than cytokine processing. It may be that a secondary canonical inflammasome, perhaps NLRP3, responds to B. pseudomallei infection only once bacterial burdens become extremely high, resulting in the observed IL-1β secretion. These observations are concordant with the work by Ceballos-Olvera et al, who additionally showed that processing of pro-IL1β to the active form was not impaired in Nlrc4−/− bone marrow-derived macrophages or in the bronchoalveolar lavage fluid of Nlrc4−/− mice infected with B. pseudomallei [24]. Furthermore, despite differences in experimental methods and timing, the histology of lungs from Nlrc4−/− mice infected with B. pseudomallei in our study appeared comparable to that of wild type mice treated with IL-1β and infected with B. pseudomallei by Ceballos-Olvera et al [24]. Notably, however, we found that systemic IL-1β and TNF-α levels were almost undetectable in Nlrc4−/− mice, despite high bacterial burdens in the spleen. This contrasted with high MIP-2 and KC concentrations in the serum, suggesting that there may be distinctly different regulatory effects of NLRC4 in various compartments.
Our data also demonstrate that NLCR4 inflammasome-dependent innate immune signaling is not the same for B. pseudomallei as other Gram-negative pulmonary pathogens. Respiratory infection with Legionella pneumophila, another Gram-negative, flagellated, intracellular pathogen is also restricted by NLRC4 and this effect is dependent on the presence of flagellin [21], [34]. However, following L. pneumophila infection there was no difference in bronchoalveolar lavage fluid cell counts or in lung cytokine levels of Nlrc4−/− mice compared to wild type mice, although there was greater histologic inflammation in the lungs of Nlrc4−/− mice [21]. As in B. pseudomallei infection, Nlrc4−/− mice are more susceptible to Klebsiella pneumoniae (a non-flagellated, extracellular pathogen) infection by the pulmonary route, with greater bacterial replication in the lungs, dissemination to the spleen, and death [35] although this effect was not observed at higher doses [36]. In contrast to our findings, pulmonary inflammation as assessed by TNF-α, KC, IL-1β, and MIP-2 levels and histologic score is reduced in Nlrc4−/− mice infected with K. pneumoniae [35]. These differences may be due to the presence of flagellin or the intracellular nature of B. pseudomallei, or to the apparent lack of NLRC4-mediated pyroptosis induced by K. pneumoniae [35].
Our human genetic study provides adjunctive evidence for the importance of NLRC4 in respiratory melioidosis although it requires validation. Few clinically associated polymorphisms in NLRC4 have been described thus far and the function of rs6757121 is otherwise unknown. We have previously reported the association of variation in TLR5 with survival in melioidosis regardless of site of infection and here show that the association holds in respiratory disease [9], [10]. Unlike in mice, modeling suggests that co-inheritance of variants in NLRC4 and in TLR5 increases the effect in an additive manner. Another important difference between mice and humans is that in humans, blunting of TLR5 function – as found in carriers of a nonsense polymorphism – is in fact protective against death from melioidosis [9], [10]. This seemingly opposite phenotype from mice underscores the challenges of mimicking human sepsis in mice [37]–[39].
In conclusion, we show that NLRC4 and TLR5 are essential elements of host defense in murine respiratory melioidosis, and that genetic variation in these genes is associated with outcome from human respiratory melioidosis.
Acknowledgments
The authors thank Loren Kinman and Tony Han for BSL-3 assistance as well as the patients and staff at Sappasithiprasong Hospital.
Author Contributions
Conceived and designed the experiments: TEW NDM HDL SJS. Performed the experiments: TEW NDM HDL. Analyzed the data: TEW. Contributed reagents/materials/analysis tools: NC WC DL VW EAM AMH SJP. Wrote the paper: TEW NC DL VW EAM AMH SJP HDL SJS.
References
- 1. Wiersinga WJ, Currie BJ, Peacock SJ (2012) Melioidosis. N Engl J Med 367: 1035–1044.
- 2. Limmathurotsakul D, Wuthiekanun V, Chierakul W, Cheng AC, Maharjan B, et al. (2005) Role and significance of quantitative urine cultures in diagnosis of melioidosis. J Clin Microbiol 43: 2274–2276.
- 3. Meumann EM, Cheng AC, Ward L, Currie BJ (2012) Clinical features and epidemiology of melioidosis pneumonia: results from a 21-year study and review of the literature. Clin Infect Dis 54: 362–369.
- 4. Kawai T, Akira S (2010) The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol 11: 373–384.
- 5. Kanneganti TD, Lamkanfi M, Nunez G (2007) Intracellular NOD-like receptors in host defense and disease. Immunity 27: 549–559.
- 6. West TE, Ernst RK, Jansson-Hutson MJ, Skerrett SJ (2008) Activation of Toll-like receptors by Burkholderia pseudomallei. BMC Immunol 9: 46.
- 7. Chantratita N, Tandhavanant S, Myers ND, Seal S, Arayawichanont A, et al. (2013) Survey of Innate Immune Responses to Burkholderia pseudomallei in Human Blood Identifies a Central Role for Lipopolysaccharide. PLoS One 8: e81617.
- 8. West TE, Chierakul W, Chantratita N, Limmathurotsakul D, Wuthiekanun V, et al. (2012) Toll-like receptor 4 region genetic variants are associated with susceptibility to melioidosis. Genes Immun 13: 38–46.
- 9. West TE, Chantratita N, Chierakul W, Limmathurotsakul D, Wuthiekanun V, et al. (2013) Impaired TLR5 Functionality Is Associated with Survival in Melioidosis. J Immunol 190: 3373–3379.
- 10. Chantratita N, Tandhavanant S, Myers ND, Chierakul W, Robertson JD, et al. (2013) Screen of whole blood responses to flagellin identifies TLR5 variation associated with outcome in melioidosis. Genes Immun 15 (2) 63–71.
- 11. Miao EA, Warren SE (2010) Innate immune detection of bacterial virulence factors via the NLRC4 inflammasome. J Clin Immunol 30: 502–506.
- 12. Bergsbaken T, Fink SL, Cookson BT (2009) Pyroptosis: host cell death and inflammation. Nat Rev Microbiol 7: 99–109.
- 13. Kofoed EM, Vance RE (2011) Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity. Nature 477: 592–595.
- 14. Zhao Y, Yang J, Shi J, Gong YN, Lu Q, et al. (2011) The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus. Nature 477: 596–600.
- 15. Yang J, Zhao Y, Shi J, Shao F (2013) Human NAIP and mouse NAIP1 recognize bacterial type III secretion needle protein for inflammasome activation. Proc Natl Acad Sci U S A 110: 14408–14413.
- 16. Rayamajhi M, Zak DE, Chavarria-Smith J, Vance RE, Miao EA (2013) Cutting edge: Mouse NAIP1 detects the type III secretion system needle protein. J Immunol 191: 3986–3989.
- 17. Vigano E, Mortellaro A (2013) Caspase-11: the driving factor for noncanonical inflammasomes. Eur J Immunol 43: 2240–2245.
- 18. Aachoui Y, Leaf IA, Hagar JA, Fontana MF, Campos CG, et al. (2013) Caspase-11 protects against bacteria that escape the vacuole. Science 339: 975–978.
- 19. West TE, Myers ND, Liggitt HD, Skerrett SJ (2012) Murine pulmonary infection and inflammation induced by inhalation of Burkholderia pseudomallei. Int J Exp Pathol 93: 421–428.
- 20. West TE, Hawn TR, Skerrett SJ (2009) Toll-like receptor signaling in airborne Burkholderia thailandensis infection. Infect Immun 77: 5612–5622.
- 21. Berrington WR, Smith KD, Skerrett SJ, Hawn TR (2013) Nucleotide-binding oligomerization domain containing-like receptor family, caspase recruitment domain (CARD) containing 4 (NLRC4) regulates intrapulmonary replication of aerosolized Legionella pneumophila. BMC Infect Dis 13: 371.
- 22. Kuida K, Lippke JA, Ku G, Harding MW, Livingston DJ, et al. (1995) Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta converting enzyme. Science 267: 2000–2003.
- 23. Wiersinga WJ, Wieland CW, Dessing MC, Chantratita N, Cheng AC, et al. (2007) Toll-Like Receptor 2 Impairs Host Defense in Gram-Negative Sepsis Caused by Burkholderia pseudomallei (Melioidosis). PLoS Med 4: e248.
- 24. Ceballos-Olvera I, Sahoo M, Miller MA, Del Barrio L, Re F (2011) Inflammasome-dependent pyroptosis and IL-18 protect against Burkholderia pseudomallei lung infection while IL-1beta is deleterious. PLoS Pathog 7: e1002452.
- 25. Breitbach K, Sun GW, Kohler J, Eske K, Wongprompitak P, et al. (2009) Caspase-1 mediates resistance in murine melioidosis. Infect Immun 77: 1589–1595.
- 26. Wiersinga WJ, Wieland CW, Roelofs JJ, van der Poll T (2008) MyD88 dependent signaling contributes to protective host defense against Burkholderia pseudomallei. PLoS ONE 3: e3494.
- 27. Sun GW, Gan YH (2010) Unraveling type III secretion systems in the highly versatile Burkholderia pseudomallei. Trends Microbiol 18: 561–568.
- 28. Stevens MP, Wood MW, Taylor LA, Monaghan P, Hawes P, et al. (2002) An Inv/Mxi-Spa-like type III protein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen. Mol Microbiol 46: 649–659.
- 29. Stevens MP, Friebel A, Taylor LA, Wood MW, Brown PJ, et al. (2003) A Burkholderia pseudomallei type III secreted protein, BopE, facilitates bacterial invasion of epithelial cells and exhibits guanine nucleotide exchange factor activity. J Bacteriol 185: 4992–4996.
- 30. Sun GW, Lu J, Pervaiz S, Cao WP, Gan YH (2005) Caspase-1 dependent macrophage death induced by Burkholderia pseudomallei. Cell Microbiol 7: 1447–1458.
- 31. Burtnick MN, Brett PJ, Nair V, Warawa JM, Woods DE, et al. (2008) Burkholderia pseudomallei type III secretion system mutants exhibit delayed vacuolar escape phenotypes in RAW 264.7 murine macrophages. Infect Immun 76: 2991–3000.
- 32. Miao EA, Mao DP, Yudkovsky N, Bonneau R, Lorang CG, et al. (2010) Innate immune detection of the type III secretion apparatus through the NLRC4 inflammasome. Proc Natl Acad Sci U S A 107: 3076–3080.
- 33. Bast A, Krause K, Schmidt IH, Pudla M, Brakopp S, et al. (2014) Caspase-1-dependent and -independent cell death pathways in Burkholderia pseudomallei infection of macrophages. PLoS Pathog 10: e1003986.
- 34. Pereira MS, Morgantetti GF, Massis LM, Horta CV, Hori JI, et al. (2011) Activation of NLRC4 by flagellated bacteria triggers caspase-1-dependent and -independent responses to restrict Legionella pneumophila replication in macrophages and in vivo. J Immunol 187: 6447–6455.
- 35. Cai S, Batra S, Wakamatsu N, Pacher P, Jeyaseelan S (2012) NLRC4 inflammasome-mediated production of IL-1beta modulates mucosal immunity in the lung against gram-negative bacterial infection. J Immunol 188: 5623–5635.
- 36. Willingham SB, Allen IC, Bergstralh DT, Brickey WJ, Huang MT, et al. (2009) NLRP3 (NALP3, Cryopyrin) facilitates in vivo caspase-1 activation, necrosis, and HMGB1 release via inflammasome-dependent and -independent pathways. J Immunol 183: 2008–2015.
- 37. Doi K, Leelahavanichkul A, Yuen PS, Star RA (2009) Animal models of sepsis and sepsis-induced kidney injury. J Clin Invest 119: 2868–2878.
- 38. Zanotti-Cavazzoni SL, Goldfarb RD (2009) Animal models of sepsis. Crit Care Clin 25: 703–viii –
- 39. Warren HS (2009) Editorial: Mouse models to study sepsis syndrome in humans. J Leukoc Biol 86: 199–201.