Role of Toll-Like Receptor 5 (TLR5) in Experimental Melioidosis

The Gram-negative intracellular pathogen Burkholderia pseudomallei is the causative agent of melioidosis, an important cause of sepsis in Southeast Asia. Recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs) is essential for an appropriate immune response during pathogen invasion. In patients with melioidosis, TLR5 is the most abundantly expressed TLR, and a hypofunctional TLR5 variant has been associated with improved survival.

ABSTRACT

The Gram-negative intracellular pathogen Burkholderia pseudomallei is the causative agent of melioidosis, an important cause of sepsis in Southeast Asia. Recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs) is essential for an appropriate immune response during pathogen invasion. In patients with melioidosis, TLR5 is the most abundantly expressed TLR, and a hypofunctional TLR5 variant has been associated with improved survival. Here, we studied the functional role of TLR5 and its ligand flagellin in experimental melioidosis. First, we observed differential TLR5 expression in the pulmonary and hepatic compartments upon infection with B. pseudomallei. Next, we found that B. pseudomallei-challenged TLR5-deficient (Tlr5^−/−) mice were more susceptible to infection than wild-type (WT) mice, as demonstrated by higher systemic bacterial loads, increased organ injury, and impaired survival. Lung bacterial loads were not different between the two groups. The phenotype was flagellin independent; no difference in in vivo virulence was observed for the flagellin-lacking mutant MM36 compared to the wild-type B. pseudomallei strain 1026b. Tlr5^−/− mice showed a similar impaired antibacterial defense when infected with MM36 or 1026b. Ex vivo experiments showed that TLR5-deficient macrophages display markedly impaired phagocytosis of B. pseudomallei. In conclusion, these data suggest that TLR5 deficiency has a detrimental flagellin-independent effect on the host response against pulmonary B. pseudomallei infection.

INTRODUCTION

Melioidosis is a severe infectious disease caused by the facultative intracellular Gram-negative pathogen Burkholderia pseudomallei, which is endemic in Southeast Asia and Northern Australia. Pneumonia is the most common disease manifestation, often accompanied by abscess formation throughout the body and sepsis (1–3). B. pseudomallei is classified as a tier 1 select agent by the Centers for Disease Control and Prevention, indicating the risk that it possesses to harm animal and human health (4). Case fatality rates for melioidosis are up to 40% despite appropriate antibiotic treatment, and even in Australia, with well-equipped health care facilities, mortality is still high (5). A recent modeling study estimated that there are 165,000 cases and 89,000 deaths annually worldwide (6). B. pseudomallei possesses an impressive arsenal of virulence factors, including the type III secretion system, lipopolysaccharide (LPS), and flagellin (3).

The importance of flagellin, essential for B. pseudomallei’s motility, in the pathogenesis of melioidosis has been highlighted by a number of studies in recent years (7–12). B. pseudomallei’s flagellum, an extracellular propeller consisting of several thousand flagellin units (13), causes upregulation of proinflammatory cytokines in mononuclear cells (8). In addition, flagella of B. pseudomallei have been implicated in cell invasion of phagocytic and nonphagocytic cells (10). Furthermore, in a mouse model of systemic infection, flagellin-deficient B. pseudomallei has been demonstrated to be less virulent than its wild-type (WT) strain (9).

Toll-like receptor 5 (TLR5) serves as the main receptor for bacterial flagellin (14, 15). In recent years, it has become clear that TLRs, which are the first receptors to detect host invasion by pathogens and initiate the immune response, play a central role in the pathogenesis of melioidosis (16–18). Previously, we reported that TLR5 expression is upregulated in both granulocytes and monocytes of melioidosis patients (16). In fact, TLR5 is the most strongly expressed TLR in whole-blood leukocytes of patients with melioidosis (19). Certain TLR5 polymorphisms are associated with impaired cytokine responses of whole blood when stimulated with B. pseudomallei flagellin (20). In addition, patients with the genetic variant TLR5_1174C>T are protected from organ damage and death, suggesting that TLR5 plays an important role in hyperinflammation during melioidosis (21). To the best of our knowledge, the in vivo interaction of flagellin and TLR5 and its effect on the host defense against B. pseudomallei infection have not been studied previously. In this study, we aimed to determine the role of TLR5 in the host response during melioidosis in vivo.

RESULTS

TLR5 deficiency facilitates bacterial growth and dissemination.

TLR5 mRNA expression is upregulated in whole-blood leukocytes of patients with melioidosis (16, 19). Correspondingly, we found that TLR5 mRNA expression was upregulated in liver tissue of B. pseudomallei-infected mice at 24 h postinfection but not in their lung tissue (see Fig. S1 in the supplemental material). We next investigated the functional role of TLR5 during experimental melioidosis and its effect on outcome. Therefore, we first infected WT and Tlr5^−/− mice with 5 × 10² CFU B. pseudomallei intranasally and observed them for 2 weeks to determine mortality (Fig. 1A). Tlr5^−/− mice showed accelerated mortality compared to WT mice (P = 0.0014), indicating that TLR5 is protective in this model of experimental melioidosis. To determine whether the increased mortality of Tlr5^−/− mice was associated with differences in bacterial loads, we performed quantitative cultures of lung (the primary site of the infection), whole blood, and liver at 24, 48, and 72 h postinfection (Fig. 1B to D). Lower pulmonary bacterial counts were seen in the Tlr5^−/− mice at 24 h postinfection, while no differences were observed in bronchoalveolar lavage fluid (BALF) (data not shown) and at later time points. However, relative to controls, Tlr5^−/− mice displayed strongly increased bacterial loads in whole blood and liver (Fig. 1C and D) at 72 h postinfection. Together, these data suggest that TLR5 plays an important role in limiting the dissemination of B. pseudomallei from the lungs to distant body sites.

FIG 1.

TLR5 deficiency impairs survival and bacterial containment. (A) Wild-type (WT) and Tlr5^−/− mice were infected with 5 × 10² CFU of B. pseudomallei 1026b intranasally and observed every 4 to 6 h during 14 days to assess survival. (B to D) B. pseudomallei-infected WT and Tlr5^−/− mice were sacrificed at 24, 48, and 72 h postinfection, and bacterial loads were determined in lung homogenates (B), blood (C), and liver (D). Data are expressed as box-and-whisker plots depicting the smallest observation, lower quartile, median, upper quartile, and largest observation (n = 20 per group for survival experiments, and n = 8 per group for bacterial quantification). ***, P < 0.001; **, P < 0.01; *, P < 0.05 (versus the WT). BC, blood culture.

TLR5 deficiency does not impact neutrophil influx and lung injury.

Intranasal inoculation with B. pseudomallei causes significant and rapid inflammation and granulocyte, but not monocyte, recruitment toward the lung (22–26). To further evaluate the role of TLR5 in the antibacterial defense against B. pseudomallei, pulmonary inflammation and granulocyte recruitment into lung tissue were assessed. B. pseudomallei infection resulted in significant pulmonary damage, as illustrated by interstitial and peribronchial inflammation, pleuritis, edema, and endothelialitis in all infected mice (Fig. 2A to C). When we compared Tlr5^−/− with WT mice, however, no differences in lung pathology were observed at 24, 48, or 72 h postinfection (Fig. 2C). Furthermore, granulocyte influx as determined by Ly6G immunostaining of lung tissue and cytometric analysis of BALF (Fig. 2D and E) as well as the presence of pulmonary myeloperoxidase (MPO) (Fig. 2F) did not differ between WT and Tlr5^−/− mice at 24 and 72 h. These results correspond with the bacterial counts in both lung and BALF at 72 h postinfection. Localized production of cytokines is important in the host defense against infection (27, 28). Therefore, we measured these mediators in the pulmonary compartment. At 24 h postinfection, diminished lung tumor necrosis factor alpha (TNF-α) and BALF keratinocyte chemoattractant (KC) levels were observed in Tlr5^−/− mice compared to controls. However, after 72 h, no differences in cytokine (TNF-α, interleukin-6 [IL-6], and IL-1β) and chemokine (KC) levels in lung homogenates or BALF of WT and Tlr5^−/− mice were observed (Table 1).

FIG 2.

Local neutrophilic influx and lung damage are not influenced by TLR5. (A and B) Representative microphotographs of H&E-stained lung tissue sections of WT (A) and Tlr5^−/− (B) mice 72 h after infection with B. pseudomallei (original magnification, ×2). (C) Lung pathology was quantified in WT and Tlr5^−/− mice infected with 5 × 10² CFU B. pseudomallei at 24, 48, and 72 h postinfection as described in Materials and Methods. (D to F) Neutrophil influx defined by Ly6G positivity, expressed as a percentage of the total lung surface (D), granulocyte influx measured by fluorescence-activated cell sorter (FACS) analysis of BALF (E), and pulmonary myeloperoxidase (MPO) activity (F). Data are expressed as box-and-whisker plots depicting the smallest observation, lower quartile, median, upper quartile, and largest observation (n = 8 mice per group). ND, not detectable.

TABLE 1.

Compartmentalized cytokine responses in lung, bronchoalveolar lavage fluid, and plasma of WT and Tlr5^−/− mice during experimental melioidosis^a

Sample type and cytokine or chemokine	Mean concn of cytokine or chemokine (pg/ml) ± SEM
	T = 24 h		T = 72 h
	WT	Tlr5−/−	WT	Tlr5−/−
Lung homogenate
TNF-α	1,261 ± 149	703 ± 158**	2,768 ± 501	2,769 ± 260
IL-6	6,085 ± 1,326	6,641 ± 1,464	6,054 ± 2,422	11,396 ± 2,719
KC	19,250 ± 2,039	13,510 ± 1,105*	22,613 ± 4,760	25,890 ± 4,372
BALF
TNF-α	1,291 ± 319	2,178 ± 652	3,449 ± 1,422	6,097 ± 1,800
IL-6	1,058 ± 140	2,722 ± 887	16,613 ± 2,971	11,050 ± 3,233
KC	4,902 ± 804	1,680 ± 217**	23,284 ± 6,781	27,880 ± 7,894
Plasma
TNF-α	7 ± 1	8 ± 1	104 ± 36	365 ± 113
IL-6	412 ± 20	316 ± 51	2,368 ± 820	6,054 ± 1,671
IL-10	BD	BD	BD	BD
MCP-1	454 ± 38	255 ± 42*	1,221 ± 405	5,465 ± 1,557
KC	11,760 ± 2,754	2,608 ± 603**	16,410 ± 6,111	21,086 ± 4,375
IFN-γ	47 ± 5	19 ± 3**	68 ± 19	1,163 ± 452**

^aShown are pulmonary, bronchoalveolar lavage fluid (BALF), and systemic cytokine levels after intranasal infection with 5 × 10² CFU wild-type B. pseudomallei 1026b. Wild-type (WT) and Tlr5^−/− mice were sacrificed 24 or 72 h after infection. Data are means ± SEM for seven or eight mice per group per time point. TNF-α, tumor necrosis factor alpha; IL, interleukin; KC, keratinocyte chemoattractant; IFN-γ, interferon gamma; BD, below the detection limit. **, P < 0.01; *, P < 0.05 (versus the WT) (Mann-Whitney U test).

TLR5 protects against systemic inflammation and liver damage.

To further evaluate the role of TLR5 in the systemic inflammatory response after infection with B. pseudomallei, we determined pro- and anti-inflammatory cytokine levels in plasma (Table 1). Twenty-four hours after infection with B. pseudomallei, we found lower plasma interferon gamma (IFN-γ), KC, and monocyte chemoattractant protein 1 (MCP-1) levels in Tlr5^−/− mice than in controls. However, at 72 h postinfection, plasma cytokine levels tended to be higher in Tlr5^−/− mice, with a significant increase of IFN-γ, most probably reflecting the increased bacterial loads in the blood (Table 1). Furthermore, we scored liver hematoxylin and eosin (H&E)-stained histology slides obtained from infected WT and Tlr5^−/− mice and performed routine clinical chemistry to evaluate hepatic and renal injury. Tlr5^−/− mice showed significantly more hepatic inflammation and damage at 72 h postinfection than did WT mice (mean histological score of 5.1 ± 0.3 versus 3.0 ± 0.4; P < 0.01) (Fig. 3A to C). Some mice showed signs of renal failure at 72 h postinfection; however, no significant differences were observed between groups. Consistent with observed liver injury, plasma levels of aspartate aminotransferase (AST) (321 ± 46 U/liter versus 88 ± 16 U/liter; P < 0.001) and alanine aminotransferase (ALT) (301 ± 30 U/liter versus 52 ± 14 U/liter; P < 0.001) were higher in Tlr5^−/− mice at 72 h postinfection than in controls (Fig. 3D and E).

FIG 3.

Increased hepatic injury in TLR5-deficient mice compared to controls in experimental melioidosis. (A and B) Representative microphotographs of H&E-stained liver tissue sections of WT (A) and Tlr5^−/− (B) mice 72 h after infection with B. pseudomallei (original magnification, ×10). The stars mark manifest necrosis. (C) Liver pathology was quantified in WT and Tlr5^−/− mice infected with 5 × 10² CFU B. pseudomallei at 24, 48, and 72 h postinfection, as described in Materials and Methods. (D and E) Plasma levels of aspartate aminotransferase (AST) (D) and alanine aminotransferase (ALAT) (E) in WT and Tlr5^−/− mice were determined 24, 48, and 72 h after infection. Data are expressed as box-and-whisker plots depicting the smallest observation, lower quartile, median, upper quartile, and largest observation (n = 8 mice per group). ***, P < 0.001; **, P < 0.01; *, P < 0.05 (versus the WT).

Protective effect of TLR5 during B. pseudomallei infection is flagellin independent.

After having confirmed that flagellin of B. pseudomallei signals through TLR5 using HEK-Blue cells stably transfected with TLR5 (Fig. 4A), we set out to determine the role of B. pseudomallei’s flagellin in our model. We first assessed the responsiveness of MH-S (murine alveolar macrophage [AM]) cells to the flagellin-lacking strain B. pseudomallei MM36 (29) and its parent strain 1026b. In MH-S cells, B. pseudomallei MM36 was slightly less potent in inducing inflammation 4 h after infection than 1026b; however, this effect disappeared after 20 h of stimulation (Fig. 4B). Similar results were found in ex vivo experiments using naive primary AM. Next, to examine if the increased susceptibility of Tlr5^−/− mice to B. pseudomallei was due to impaired flagellin recognition, we infected WT and Tlr5^−/− mice with 5 × 10² CFU of the flagellin-lacking B. pseudomallei strain MM36. At predefined time points, we sacrificed mice to investigate lung, whole-blood, and liver bacterial counts (Fig. 4C to E). Similar to our above-described experiments with the wild-type strain B. pseudomallei 1026b, Tlr5^−/− mice infected with B. pseudomallei MM36 demonstrated higher bacterial loads in whole blood and liver than did controls. In addition, we determined cytokine levels in plasma, lung homogenates, and BALF (Table 2). No differences were found in lung and BALF TNF-α and IL-6 levels. Pulmonary neutrophil-attracting KC levels were significantly lower at 24 h postinfection in Tlr5^−/− mice. In plasma, IFN-γ and MCP-1 levels were significantly higher in Tlr5^−/− mice 72 h after infection with B. pseudomallei MM36 than in controls, which is in line with our observations in mice infected with B. pseudomallei 1026b. To assess the local and systemic inflammatory response further, lung and liver pathology was scored (Fig. 4F and G), and we again found that, similar to experiments using wild-type B. pseudomallei, liver pathology was more severe in Tlr5^−/− mice than in WT mice. In concordance with the increased liver pathology, plasma levels of AST (357 ± 66 U/liter versus 90 ± 11 U/liter; P < 0.05) and ALT (340 ± 58 U/liter versus 47 ± 5 U/liter; P < 0.05) were higher in Tlr5^−/− mice at 72 h postinfection (Fig. 4H and I). Taken together, these results remarkably demonstrate that the observed protective effect of TLR5 in this murine model of melioidosis is independent of B. pseudomallei’s flagellin.

FIG 4.

TLR5’s protective role during experimental melioidosis is flagellin independent. (A) HEK-Blue cells stably transfected with TLR5 were stimulated with medium, LPS of E. coli (100 ng/ml), or increasing concentrations (1, 10, or 100 ng/ml) of flagellin of S. Typhimurium or B. pseudomallei (B. ps) 1026b for 20 h, after which the optical density at 655 nm was measured. (B) The murine alveolar macrophage cell line MH-S was stimulated with medium, LPS of E. coli (100 ng/ml), or heat-killed B. pseudomallei 1026b or MM36 (10⁷ CFU/ml) for 4 and 20 h, after which TNF-α levels were measured. Data are represented as means ± standard errors of the means (SEM) (n = 4 per group). (C to E) WT and Tlr5^−/− mice were intranasally infected with 5 × 10² CFU B. pseudomallei MM36, a flagellin-lacking mutant, and sacrificed at 24, 48, or 72 h postinfection. Bacterial loads were determined in lung homogenates (C), blood (D), and liver (E). (F and G) Lung (F) and liver (G) pathology was scored as described in Materials and Methods. (H and I) Plasma AST (H) and ALT (I) levels were measured at 24, 48, and 72 h postinfection. Data are expressed as box-and-whisker plots depicting the smallest observation, lower quartile, median, upper quartile, and largest observation (n = 8 mice per group). ***, P < 0.001; **, P < 0.01; *, P < 0.05 (versus the WT). BC, blood culture.

TABLE 2.

Compartmentalized cytokine responses in lung, bronchoalveolar lavage fluid, and plasma of WT and Tlr5^−/− mice after infection with flagellin-lacking B. pseudomallei strain MM36^a

Sample and cytokine or chemokine	Mean concn of cytokine or chemokine ± SEM
	T = 24 h		T = 72 h
	WT	Tlr5−/−	WT	Tlr5−/−
Lung homogenate
TNF-α	1,328 ± 142	954 ± 180	4,820 ± 1,205	3,837 ± 629
IL-6	10,030 ± 3,071	8,668 ± 1,513	12,489 ± 6,622	31,125 ± 8,799
KC	12,634 ± 1,460	2,742 ± 401***	15,669 ± 6,339	3,638 ± 1,319
BALF
TNF-α	729 ± 127	993 ± 138	10,613 ± 3,066	11,694 ± 3,199
IL-6	925 ± 172	1,373 ± 211	20,890 ± 8,197	29,861 ± 8,972
KC	1,433 ± 513	535 ± 138	20,503 ± 12,038	16,984 ± 7,700
Plasma
TNF-α	5 ± 1	3 ± 0*	76 ± 28	305 ± 119
IL-6	253 ± 75	213 ± 65	2,246 ± 973	4,404 ± 1,602
IL-10	ND	ND	ND	ND
MCP-1	368 ± 28	187 ± 22	739 ± 300	4,323 ± 1,537*
IFN-γ	30 ± 5	15 ± 2*	31 ± 9	301 ± 106*

^aShown are pulmonary, bronchoalveolar lavage fluid (BALF), and systemic cytokine levels after intranasal infection with 5 × 10² CFU of the flagellum-lacking mutant B. pseudomallei MM36. Wild-type (WT) and Tlr5^−/− mice were sacrificed 24 or 72 h after infection. Data are means ± SEM for seven or eight mice per group per time point. TNF-α, tumor necrosis factor alpha; IL, interleukin; KC, keratinocyte chemoattractant; IFN-γ, interferon gamma; ND, not detectable. *, P < 0.05; ***, P < 0.001 (Mann-Whitney U test).

Impaired phagocytosis of B. pseudomallei in TLR5-deficient macrophages.

The early host response to B. pseudomallei infection is characterized by a coordinated series of effector functions, which include cytokine production and killing and phagocytosis of the invading agent (2, 3). To obtain additional insights into the function of TLR5 in the host defense against B. pseudomallei, we analyzed the requirement of TLR5 for cytokine production of isolated whole-blood leukocytes, AM, and bone marrow-derived macrophages (BMDM) upon stimulation with B. pseudomallei. Stimulation of whole blood of Tlr5^−/− mice with B. pseudomallei led to increased levels of proinflammatory cytokines, such as TNF-α (Fig. 5A), compared to controls. Flagellin was only a weak stimulus of whole blood. In contrast, TLR5-deficient AM and BMDM released equal amounts of TNF-α upon 4 or 20 h of stimulation with B. pseudomallei compared to WT cells (Fig. 5B and C). Having thus found that TLR5 only partially contributes to cellular responsiveness to B. pseudomallei in vitro (Fig. 4B and Fig. 5B and C), we finally wished to determine if TLR5 plays a role in killing and/or phagocytosis of B. pseudomallei. Both WT and Tlr5^−/− macrophages effectively killed >99% of B. pseudomallei bacteria (Fig. S2A). However, Tlr5^−/− macrophages demonstrated a markedly diminished capacity to phagocytose B. pseudomallei (Fig. 5D).

FIG 5.

Decreased phagocytosis of TLR5-deficient BMDM. (A to C) Whole blood (A), alveolar macrophages (AM) (B), and bone marrow-derived macrophages (BMDM) (C) of WT and Tlr5^−/− mice were stimulated with medium, flagellin of S. Typhimurium (100 ng/ml), or heat-killed wild-type B. pseudomallei 1026b (10⁷ CFU/ml or an MOI of 50). The supernatant was collected after 20 h of stimulation and assayed for mTNF-α. (D) BMDM of WT and Tlr5^−/−mice were incubated at 37°C with carboxyfluorescein N-hydroxysuccinimidyl ester (CFSE)-labeled heat-killed B. pseudomallei 1026b, after which phagocytosis was determined at 30 and 60 min, as described in Materials and Methods. Data are expressed as box-and-whisker plots depicting the smallest observation, lower quartile, median, upper quartile, and largest observation (n = 4 mice per group). **, P < 0.01; *, P < 0.05 (versus the WT).

DISCUSSION

In this study, we show that TLR5 deficiency leads to increased bacterial loads, distant organ damage, and impaired survival in experimental melioidosis. Of note, susceptibility to B. pseudomallei was flagellin independent, as demonstrated by the results obtained when WT and Tlr5^−/− mice were infected with the flagellin-lacking strain MM36. Further ex vivo experiments showed that Tlr5^−/− macrophages display markedly impaired phagocytosis of B. pseudomallei. These data are the first to demonstrate the functional in vivo role of TLR5 during melioidosis in terms of bacterial dissemination and inflammation.

TLR5 is the 25th most upregulated gene of all 48,803 genes that were analyzed in the event of melioidosis (19). Correspondingly, protein expression data show that TLR5 is the most abundantly expressed TLR in patients with melioidosis, even more so than TLR2 and TLR4 (16). To elucidate the role of TLR5 in the induction of antibacterial host defense, we used our well-established murine model of pneumonia-derived melioidosis (16, 22, 26, 30) and found that Tlr5^−/− mice were significantly more susceptible to B. pseudomallei infection, as reflected by higher mortality rates, increased systemic bacterial loads, and more severe distant organ damage. TLR5 has previously been shown to be involved in neutrophil recruitment in the lungs and the local pulmonary cytokine response to flagellin and flagellated bacteria (28, 31–33); however, we did not find an impaired neutrophilic influx in Tlr5^−/− mice upon infection with either B. pseudomallei 1026b or MM36.

We confirmed that flagellin of B. pseudomallei signals via TLR5 by stimulating HEK-TLR5 cells with flagellin of B. pseudomallei. Furthermore, our study sheds new light upon the ongoing debate of flagellin as a virulence factor of B. pseudomallei (9, 10, 29, 34). In our model of pneumonia-derived melioidosis, we did not observe differences in bacterial counts or inflammation when we compared WT mice infected with B. pseudomallei 1026b to those infected with the flagellin-lacking strain MM36. Similarly, we did not notice differences in the cellular responsiveness of MH-S cells, AM, or BMDM after stimulation with both strains (Fig. 4B; see also Fig. S2B and C in the supplemental material). In contrast, previous reports found diminished virulence of flagellin mutants compared to their parent strains (7–11). Differences in the B. pseudomallei parent strains, mouse strains, and infection models should be taken into account when comparing these results. Finally, it should be noted that, in addition to TLR5, flagellin can also be recognized by the NOD-like receptor NLRC4 (15, 35). NLRC4 can recognize cytoplasmically delivered flagellin and has been associated with the regulation of pyroptosis in experimental melioidosis (36). TLR5 and NLRC4 expressions are induced as part of the type I interferon and TNF-α responses. Both pathways are prominent in the host response to melioidosis, as evidenced by previous work demonstrating that mice lacking TLR5 or NLRC4 display increased mortality in experimental melioidosis (19, 23, 37). The presence of NLRC4 might in part explain why the observed phenotype of Tlr5^−/− mice is flagellin independent.

Since macrophages play a central role in any attempt to successfully clear an infection with B. pseudomallei (38, 39), we investigated the impact of TLR5 deficiency on its cellular functions. Although TLR5 did not play a major role in the in vitro cellular responsiveness of murine alveolar macrophages and BMDMs toward B. pseudomallei, we found that Tlr5^−/− macrophages were significantly less capable of phagocytosing B. pseudomallei than WT cells. This is in concurrence with the work of Descamps et al., who demonstrated that phagocytosis of Pseudomonas aeruginosa by Tlr5^−/− macrophages is impaired (40).

It has been described that Tlr5^−/− mice can have a different composition of the gut microbiota (41–43) and that vertical (parental) transmission and diet are major determinants of gut microbiota composition (44, 45). Analyses of the gut microbiota of naive Tlr5^−/− mice and WT controls by using IS-pro platform technology (46) indeed showed that uninfected Tlr5^−/− mice exhibit a different microbiota composition than uninfected WT mice (Fig. S3). In Tlr5^−/− mice, an increase in Proteobacteria was seen, as was a decrease in Firmicutes. The basal inflammatory phenotype was not different between WT and Tlr5^−/− mice: body weight, colon weight, and serum amyloid A levels did not differ between strains, which is in line with a previous report (47). Familial transmission associated with long-term breeding of isolated mouse colonies rather than defective innate immunity most likely accounts for these marked compositional differences in gut microbiota (43). In addition, we have recently identified the gut microbiota as a potential modulator of innate immunity during murine B. pseudomallei infection (48). To investigate whether the role of the gut microbiome can explain the observed phenotype, further experiments using gnotobiotic mice will be required.

Limitations need to be considered. First, caution is needed when extrapolating these data derived from mouse experiments to human disease. Human carriers of a hyporesponsive TLR5 polymorphism are in part protected against death from melioidosis (21, 49). In this respect, it is of interest that TLR5 hyporesponsiveness in humans leads to higher susceptibility to invasive aspergillosis (50), recurrent urinary tract infections (51), and Legionnaires’ disease (52). Second, murine models like the one used here make use of a homogenous group of experimental animals, in terms of sex and (relatively young) age, exposed to a well-controlled bacterial challenge, whereas patients form a heterogeneous group in which multiple factors modify disease outcome, including the extent of pathogen exposure, older age, comorbidities, comedications, and genetic composition. Third, ex vivo, we demonstrated impaired phagocytosis in Tlr5^−/− mice; however, the significance of this finding in vivo remains to be elucidated. Despite these caveats, the present study now describes the potential role of TLR5 in the host response in a clinically relevant model of melioidosis.

In conclusion, these data suggest that TLR5 deficiency has a detrimental flagellin-independent effect on the host response against pulmonary B. pseudomallei infection. Interventions to modulate TLR5 function are being designed for experimental pneumonia (53) and may represent a target for immunomodulation in septic melioidosis patients.

MATERIALS AND METHODS

Mice and ethics statement.

TLR5-deficient (Tlr5^−/−) mice were provided by Richard Flavell (Yale University School of Medicine, New Haven, CT) and were backcrossed at least eight times on a C57BL/6 genetic background (32). Age- and sex-matched specific-pathogen-free 8- to 10-week-old WT C57BL/6 mice were purchased from Charles River. The animals were fed the same diet. The Institutional Animal Care and Use Committee of the Academic Medical Center approved all experiments (permit number DIX#102370). Experiments were carried out in accordance with the Dutch Experiments on Animals Act.

Experimental infection and determination of bacterial growth.

Experimental melioidosis was induced by intranasal inoculation with 5 × 10² CFU/50 μl B. pseudomallei strain 1026b or flagellin-lacking strain MM36 (from parent strain 1026b) (29), as previously described (22, 54, 55). For survival experiments, mice were monitored for 14 days and checked every 6 h until death occurred. Sample harvesting, processing, and determination of bacterial growth were performed as described previously (22, 30, 54). Bronchoalveolar lavage (BAL) was performed by instilling two 0.5-ml aliquots of sterile saline into the trachea (22, 30). Lung, spleen, and liver were harvested and homogenized in 4 volumes of sterile isotonic saline. For bacterial quantification, blood, BALF, and organ homogenates were serially diluted 10-fold in sterile isotonic saline and plated onto sheep blood agar plates. Following overnight incubation at 37°C with 5% CO₂, CFU were counted. For cytokine measurements, homogenates were diluted 1:1 with lysis buffer (300 mM NaCl, 30 mM Tris, 2 mM MgCl₂, 2 mM CaCl₂, 1% [vol/vol] Triton X-100 [pH 7.4]) with a protease inhibitor mix (Complete protease inhibitor cocktail tablets; Roche) and incubated for 30 min on ice, followed by centrifugation at 680 × g for 10 min. Supernatants were stored at −20°C until analyses. Pulmonary cell suspensions obtained from infected mice were evaluated by fluorescence-activated cell sorter (FACS) analysis (BD Biosciences), as described previously (22, 30).

Evaluation of TLR5 mRNA levels by RT-PCR.

Reverse transcriptase PCRs (RT-PCRs) were performed on cDNA samples that were 4-fold diluted in H₂O using FastStart DNA master SYBR green I (Roche) in a LightCycler apparatus (Roche). The primers used for murine TLR5 were 5′-ACCACACTTCAGCAGGATCA-3′ and 5′-ATCCAGGGAATCTGGGTGA-3′ (56). PCR conditions were as follows: 5 min at 95°C and 40 cycles of amplification (95°C for 15 s, 60°C for 5 s, and 72°C for 20 s). For quantification, standard curves were constructed by PCR on serial dilutions of concentrated cDNA; data were analyzed using LightCycler software as described by the manufacturer. Lung and liver total RNA of mice was isolated using an Isolate II RNA minikit (Bioline), treated with RNase-free DNase (Bioline), and reverse transcribed using an oligo(dT) primer and Moloney murine leukemia virus RT (Promega), in accordance with the manufacturers’ recommendations. Gene expression is presented as the ratio of TLR5 expression to the expression of the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT).

Assays.

Plasma mouse tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-10, monocyte chemoattractant protein 1 (MCP-1), and interferon gamma (IFN-γ) were measured by a cytometric bead array multiplex assay (BD Biosciences). Pulmonary and BALF keratinocyte chemoattractant (KC), TNF-α, IL-6, IL-1β, and myeloperoxidase (MPO) levels were measured by an enzyme-linked immunosorbent assay (ELISA) (R&D Systems). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were determined with Cobas 8000 module c702 (Roche Diagnostics) in accordance with the manufacturer’s recommendations.

Histology and immunohistology.

Lungs, livers, and spleens for histology were harvested after infection, fixed in 100% formalin, and embedded in paraffin. Sections of 4 μm were stained with hematoxylin and eosin and analyzed as described previously (16, 57). Lungs were scored for affected surface, necrosis and/or formation of an abscess, interstitial inflammation, endothelialitis, bronchitis, edema, thrombus formation, and pleuritis on a scale of 0 (absent) to 4 (most severe). The maximum lung pathology score was 32. Spleen and liver sections were scored for inflammation, necrosis/abscess formation, and thrombus formation using the scale described above. The maximum total spleen or liver inflammation score was 12. Granulocyte (Ly6G) staining, using fluorescein isothiocyanate (FITC)-labeled rat anti-mouse Ly6G monoclonal antibody (mAb) (BD Pharmingen), was done exactly as described previously (58). Stained areas were analyzed with ImageJ (version 2006.02.01; U.S. National Institutes of Health) and expressed as a percentage of the total lung surface area.

Preparation of alveolar macrophages and bone marrow-derived macrophages.

Alveolar macrophages (AM) were harvested from WT and Tlr5^−/− mice by BAL as described previously (16) and seeded at a final concentration of 2 × 10⁴ cells/100 μl. Cells were cultured in 96-well microtiter plates at 37°C in 5% CO₂ air overnight; washed with RPMI 1640 (Life Technologies, Bleiswijk, The Netherlands) containing 1 mM pyruvate, 2 mM l-glutamine, penicillin-streptomycin (100 U penicillin, 100 g streptomycin), and 10% fetal calf serum (FCS) (Life Technologies) at a final concentration of 2 × 10⁴ cells/100 μl; and seeded in 96-well flat-bottom plates (Greiner Bio-One) to remove nonadherent cells, before AM were used for further assays. Bone marrow-derived macrophages (BMDM) were harvested as described previously (30, 59). In short, tibias and femurs were aseptically removed and flushed with phosphate-buffered saline (PBS). The collected BMDM were cultured for 10 days in serum-free medium, before being seeded at the required concentration.

Cellular function assays.

Stimulation assays were performed using whole blood, AM, and BMDM of WT and Tlr5^−/− mice (n = 8). Whole blood and isolated cells were stimulated with medium (RPMI 1640), flagellin of Salmonella enterica serovar Typhimurium (100 ng/ml) (InvivoGen, San Diego, CA, USA), or heat-killed B. pseudomallei strain 1026b (10⁷ CFU/ml). After 20 h at 37°C in 5% CO₂ air, plates were centrifuged at 1,250 rpm for 5 min, and supernatants were harvested and stored at −20°C until assayed for murine TNF-α (mTNF-α) and IL-6. For additional cytokine release assays, the murine alveolar macrophage cell line MH-S (ATCC, Manassas, VA, USA) was used, and cells were seeded at a concentration of 10⁶ cells/ml in a 96-well plate and stimulated with medium, Escherichia coli LPS (InvivoGen) (100 ng/ml), or heat-killed B. pseudomallei 1026b or MM36 (10⁷ CFU/ml) for 4 and 20 h. The harvested supernatants were stored at −20°C until assayed for mTNF-α.

Intracellular killing and phagocytosis.

To assess intracellular killing, BMDM were seeded in 96-well plates at 1 × 10⁵ cells/100 μl and left to attach for 3 h at 37°C in 5% CO₂ air. BMDM were then washed with medium and stimulated overnight with E. coli LPS (100 ng/ml). The following day, BMDM were washed twice with fresh medium and infected with B. pseudomallei 1026b bacteria grown to log phase (multiplicity of infection [MOI] of 40) for 20 min at 37°C in 5% CO₂ air, after which they were washed twice again and incubated with kanamycin at 250 μg/ml for 30 min at 37°C in 5% CO₂ air (36). This was time zero. BMDM were washed twice with sterile PBS and subsequently lysed with 0.1% Triton X-100 (Sigma-Aldrich) for 5 min at selected time points. Appropriate dilutions of these lysates were plated onto blood agar plates and incubated at 37°C for 24 to 48 h before colony counts were performed.

Phagocytosis was performed as described previously (54). In short, BMDM (5 × 10⁴ cells/well) were stimulated with heat-killed, FITC-labeled B. pseudomallei bacteria (MOI of 100) for 30 and 60 min at 37°C, after which they were washed twice with ice-cold 1× PBS. BMDM were then suspended in 100 μl FACS buffer and quenched with 50 μl trypan blue for 1 min, followed by washing with FACS buffer. Samples were analyzed directly after sample collection by flow cytometry using FlowJo. Control wells were kept at 4°C, and their values were subtracted from the sample values.

Human embryonic kidney cells.

Human embryonic kidney HEK-Blue cells stably expressing murine TLR5 (HEK-Blue-TLR5) (InvivoGen) and their naked parent cell line HEK-Blue-Null were cultured, according to the manufacturer’s instructions, in Dulbecco’s modified Eagle’s medium (DMEM) plus 4.5 g/liter glucose (Life Technologies), 10% FCS, 50 U/ml penicillin, 50 mg/ml streptomycin, 100 mg/ml Normocin, and 2 mM l-glutamine. Cells were stimulated with medium, flagellin of S. Typhimurium (1 to 100 ng/ml), or flagellin of B. pseudomallei 1026b (1 to 100 ng/ml) (kindly provided by Donald E. Woods, University of Calgary, Alberta, Canada). In selected experiments, HEK-Blue-Null and HEK-Blue-TLR5 cells were incubated with plasma of mice infected with B. pseudomallei 1026b or MM36 to assess the presence of circulating TLR5 ligands in the systemic compartment during experimental melioidosis. At 20 h poststimulation, levels of secreted embryonic alkaline phosphatase (SEAP) were determined by spectrophotometry at 650 nm.

Statistical analysis.

Data are expressed as box-and-whisker plots or as bars (medians with ranges or means with standard deviations [SD], respectively). Comparisons between groups were first performed using one-way analysis of variance on ranks (ANOVA); only when significant differences were present were groups at individual time points tested using the Mann-Whitney U test. Survival rates were compared using the Kaplan-Meier method, followed by the log rank test. Analyses were done using GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA). P values of <0.05 were considered statistically significant.