Triggering Receptor Expressed on Myeloid Cells (TREM)-2 Impairs Host Defense in Experimental Melioidosis

Background Triggering receptor expressed on myeloid cells (TREM) -1 and TREM-2 are key regulators of the inflammatory response that are involved in the clearance of invading pathogens. Melioidosis, caused by the "Tier 1" biothreat agent Burkholderia pseudomallei, is a common form of community-acquired sepsis in Southeast-Asia. TREM-1 has been suggested as a biomarker for sepsis and melioidosis. We aimed to characterize the expression and function of TREM-1 and TREM-2 in melioidosis. Methodology/Principal Findings Wild-type, TREM-1/3 (Trem-1/3-/-) and TREM-2 (Trem-2-/-) deficient mice were intranasally infected with live B. pseudomallei and killed after 24, and/or 72 h for the harvesting of lungs, liver, spleen, and blood. Additionally, survival studies were performed. Cellular functions were further analyzed by stimulation and/or infection of isolated cells. TREM-1 and TREM-2 expression was increased both in the lung and liver of B. pseudomallei-infected mice. Strikingly, Trem-2-/-, but not Trem-1/3-/-, mice displayed a markedly improved host defense as reflected by a strong survival advantage together with decreased bacterial loads, less inflammation and reduced organ injury. Cellular responsiveness of TREM-2, but not TREM-1, deficient blood and bone-marrow derived macrophages (BMDM) was diminished upon exposure to B. pseudomallei. Phagocytosis and intracellular killing of B. pseudomallei by BMDM and alveolar macrophages were TREM-1 and TREM-2-independent. Conclusions/Significance We found that TREM-2, and to a lesser extent TREM-1, plays a remarkable detrimental role in the host defense against a clinically relevant Gram-negative pathogen in mice: TREM-2 deficiency restricts the inflammatory response, thereby decreasing organ damage and mortality.


Introduction
In sepsis, defined as a deregulated host response to a life-threatening infection, a careful balance between inflammatory and anti-inflammatory responses is vital [1][2][3]. Pathogen-or danger-associated molecular patterns are recognized by intracellular sensory complexes and cell surface receptors expressed on innate immune cells that can initiate the inflammatory and anti-microbial response. Well-known examples of these pattern recognition receptors (PRRs) are the Toll-like receptor (TLR), nucleotide-oligomerization domain-like receptor (NLR) and C-type lectin receptor (CLR) families [4]. A more recently discovered group of innate immune receptors are the membrane-bound triggering receptors expressed on myeloid cells (TREMs), which act as key modulators, rather than as initiators, of the inflammatory response [5][6][7].
TREM-1 and TREM-2 are the most studied members of the TREM-family, however their exact role in the pathogenesis of sepsis remains ill-defined. Upon recognition of partially still unspecified ligands, both receptors phosphorylate the adaptor molecule DNAX adaptor protein 12 (DAP12) after which the cellular response is initiated [8,9]. Only recently, binding of TREM-1 to a complex of peptidoglycan recognition protein 1 (PGLYRP1) and bacterially derived peptidoglycan has been demonstrated [10]. TREM-1 is expressed on neutrophils and monocyte subsets [11] and amplifies pro-inflammatory TLR-mediated responses in vitro [12]. There are conflicting reports on the role of TREM-1 in in vivo infection models. TREM-1 deficiency impaired bacterial clearance in a model of Klebsiella pneumonia-induced liver abscess formation [13], pneumococcal [14] and Pseudomonas (P.) aeruginosa pneumonia [15]. However, blocking TREM-1 with an analogue synthetic peptide derived from the extracellular moiety of TREM-1 (LP17) actually improved survival during gram-negative sepsis [16] and endotoxaemia [17]. Interestingly, in a murine pneumonia model of Legionella pneumonia no impact of TREM-1 deficiency was found on bacterial clearance or neutrophil influx towards the primary site of infection [18].
Melioidosis, considered to be an illustrative model for Gram-negative sepsis, is caused by the Tier 1 biological treat agent Burkholderia pseudomallei [28,29]. Melioidosis is characterized by pneumonia and abscess formation and an important cause of community-acquired sepsis in Southeast Asia and Northern Australia [28]. The high mortality rate, that can approach 40%, and the emerging antibiotic resistance of B.pseudomallei [30] emphasize the need to better understand the pathogenesis of melioidosis, which could ultimately lead to novel treatment strategies. We previously found increased soluble (s) TREM-1 plasma levels and TREM-1 surface expression on monocytes of patients with melioidosis [31], suggesting an important role for TREM-1 in the host defense against B. pseudomallei. Treatment with a peptide mimicking a conserveddomain of sTREM-1 partially protected mice from B. pseudomallei induced lethality [31].
In this study we now examine the role of TREM-1 and TREM-2 during experimental melioidosis, utilizing recently generated Trem-1/3-deficient (Trem-1/3 -/-) [15] and Trem-2deficient (Trem-2 -/-) mice [19] to determine their contribution to the host response against B. pseudomallei. We hypothesized that TREM-1 deficiency would decrease inflammation and improve survival during murine melioidosis while TREM-2 deficiency would instead lead towards increased inflammation and a worsened survival. Unexpectedly however, we found that TREM-2, but not TREM-1, plays an important detrimental role during melioidosis. TREM-2 deficiency improves survival of B. pseudomallei infected mice, by limiting inflammation and organ damage. These data identify TREM-2 as a potential treatment target for sepsis caused by B. pseudomallei.

Materials and Methods
Detailed methods are provided in the online supplement (S1 Appendix).

Ethics statement
The Animal Care and Use of Committee of the University of Amsterdam approved all experiments (DIX102273), which adhered to European legislation (Directive 2010/63/EU).

Experimental infection and assays
B. pseudomallei, derived from our aliquoted frozen stock, was grown to log-phase and further diluted in sterile PBS (1x). Experimental melioidosis was induced by intranasal inoculation with 5 × 102 colony forming units (CFU) of B. pseudomallei strain 1026b (a clinical isolate) as described [32][33][34]. For survival experiments mice were observed 4-6 times daily, up to 14 days post-infection. Sample harvesting, processing, and determination of bacterial growth were performed as described in detail in the S1 Appendix [33,34]. All work concerning live B. pseudomallei was performed in a (A)BSL III facility.
Chemo-and cytokine levels were determined in plasma, lung and liver. Distant organ damage was more closely assessed by plasma transaminases, lactate dehydrogenase (LDH) and blood urea nitrogen (BUN) levels.

TREM-1 and TREM-2 expression
Total RNA was isolated using the Isolate II RNA mini kit (Bioline, Taunton, MA, USA), treated with DNase (Bioline) and reverse transcribed using an oligo(dT) primer and Moloney murine leukemia virus RT (Promega, Madison, WI, USA). Primers and RT-PCR conditions can be found in the supplemental data. Data were analyzed using the comparative Ct method.

Phagocytosis and bacterial killing
Phagocytosis was determined as described previously [38]. In brief, AM and BMDM (5x 104 cells/well) were incubated with or without heat-inactivated FITC-labelled B. pseudomallei (MOI 50) for 60 and/or 120 minutes at 37°C and 5% CO2 air and internalization was measured directly after collection by flow cytometry.
Bacterial killing was evaluated as described [36,39]. In short, BMDM were incubated with to log-phase grown B. pseudomallei (MOI 30) for 20 minutes at 37°C in 5% CO2 air, after which they were washed and incubated with kanamycin 250 μg/ml for 30 minutes at 37°C in 5% CO2 air (this point was taken as time zero) [36]. At designated time points the BMDM were washed and lysed and appropriate dilutions of these lysates were plated onto blood-agar plates and incubated at 37°C for 24-48 h before CFU were counted.

Results
Increased TREM-1 and TREM-2 expression in the lung and liver during experimental melioidosis Septic melioidosis patients present with pneumonia and bacterial dissemination to distant body sites [28,40]. Since it is not feasible to study TREM-1 and TREM-2 mRNA expression at tissue level in these patients, we used our well-established murine model of pneumonia-derived melioidosis in which mice are intranasally infected with a lethal dose of B. pseudomallei [33,34]. Mice were killed at 0, 24, and 72h after infection (i.e., directly before the first predicted death), and TREM-1/-2 mRNA expression was determined in lungs and livers. At baseline, TREM-1 and TREM-2 expression was low, corresponding with our previous data on sTREM-1 levels in melioidosis patients [31], TREM-1 was strongly up-regulated in lung and liver tissue (P<0.05 lung at 24h, P<0.01 liver at 72h; Fig 1A and 1B). TREM-2 mRNA expression was increased in experimental melioidosis as well (P<0.05 in both lung and liver; Fig 1C and 1D). The increase in both TREM-1 and TREM-2 expression was much more pronounced at the primary site of infection, the pulmonary compartment, when compared to the hepatic compartment.

pseudomallei-induced mortality
Having established that both TREM-1 and TREM-2 are highly up-regulated during melioidosis, we further investigated the involvement of these receptors in the outcome of melioidosis. Therefore, we infected WT, Trem-1/3 -/and Trem-2 -/mice intranasally with a lethal dose of B. pseudomallei and observed them for 14 days (Fig 2A and 2B). There was no significant difference in survival between Trem-1/3 -/and WT mice following a lethal B. pseudomallei challenge: 95% of Trem-1/3 -/and WT mice died within 6 days after inoculation (Fig 2A). Strikingly however, Trem-2 -/mice were significantly protected: 70% of Trem-2 -/survived until the end of the 14-day observation period while all WT mice died within 6 days (P< 0.001; Fig 2B).

Enhanced bacterial clearance in Trem-2 -/mice
To substantiate the finding that Trem-2 -/mice are protected during melioidosis, we determined bacterial loads in lung and BALF as well as in blood, liver and spleen 72h post-infection. Relative to WT mice, Trem2 -/mice displayed strongly reduced bacterial loads both at the

Trem-2 -/mice demonstrate reduced lung inflammation
Since TREM-2 has been described as a negative regulator of inflammation [19,20], we next assessed the inflammatory response in the pulmonary compartment. Therefore we studied the extent of inflammation in lung homogenates and BALF. We observed markedly decreased levels of pro-inflammatory cytokines TNF-α, IL-6, IL-1β and the chemokine KC in both lung homogenates and BALF of TREM-2 deficient mice compared to controls (P<0.01-0.05; Table 1). To further obtain insight into the involvement of TREM-2 in the inflammatory response following B. pseudomallei infection, we semi-quantitatively scored lung histology slides generated from Trem-2 -/and WT mice. However, all mice displayed severe pulmonary inflammation and no differences were observed between the mouse strains (Fig 3A-3C). Neutrophil recruitment to the lung is an essential part of the inflammatory host response to melioidosis. Therefore, we determined the granulocyte influx into the pulmonary compartment by Ly6G-immunostaining in WT and Trem-2 -/mice 72h post-infection with B. pseudomallei (Fig 3D-3F). This immunostaining recognizes Gr-1, that is granulocyte-specific, Corresponding to the diminished bacterial loads and decreased levels of cyto-and chemokines in lung tissue, a reduced influx of granulocytes in lungs of Trem-2 -/mice was found (P<0.05, Fig 3D).

Trem-2 deficiency leads to decreased distant organ injury during experimental melioidosis
To evaluate the role of TREM-2 in the systemic inflammatory response, we determined plasma cytokine levels 72h post-infection with B. pseudomallei. Consistent with the lower pulmonary cytokine levels and bacterial loads, we found that the plasma levels of TNF-α, IL-6, IL-1β, MCP-1, IL-10, IFN-γ and KC were all significantly reduced in Trem-2 -/mice compared to WTs (P<0.01-0.05, Table 1). Furthermore, we obtained spleen pathology scores and performed routine clinical chemistry tests to evaluate hepatic, renal and systemic injury. In line with the observed decreased splenic bacterial loads, Trem-2 -/mice showed less inflammation compared to WT mice 72h after inoculation with B. pseudomallei (P<0.05; Fig 4A). Plasma AST levels of Trem-2 -/mice were decreased when compared to controls 72h post-infection, reflecting decreased hepatocellular injury in these animals (P<0.05; Fig 4B). Consistently, we  observed a trend towards lower ALT, BUN and LDH levels in Trem-2 -/mice compared to controls suggesting less organ damage respectively (Fig 4C-4E).
Lack of TREM-2 leads to a reduced inflammatory response ex vivo, but does not impact on phagocytosis of B. pseudomallei by macrophages Having established that TREM-2 plays an important deleterious role during experimental melioidosis and is involved in the inflammatory response, we next assessed what cells are responsible for these effects. It is known that blood monocytes, alveolar macrophages (AM) and BMDM express TREM-2 [25], therefore we harvested these cells and first stimulated them overnight with the TLR4-ligand LPS and B. pseudomallei. We found a clear trend towards The Role of TREM-1 and -2 in Melioidosis lower TNF-α levels when whole blood, AM or BMDM of Trem-2 -/mice were stimulated with LPS (Fig 5A-5C). This effect was even more pronounced after stimulation with B. pseudomallei: the TNF-α response of whole blood and BMDM derived from TREM-2 deficient mice was significantly reduced compared to controls (P<0.05; Fig 5A and 5B). Considering TREM-2's known phagocytic properties [24,25] and the observed lower local and systemic bacterial loads in TREM-2-deficient mice, we determined the phagocytic capacity of AM and BMDM harvested from WT and Trem-2 -/mice. Despite a trend towards enhanced phagocytosis of FITClabelled B. pseudomallei by TREM-2 deficient macrophages, no significant differences were found (Fig 5D and 5E). In line, TREM-2 did not impact on the intracellular killing of B. pseudomallei by BMDM (S1 Fig).

Limited role of TREM-1/3 in the host defense during experimental melioidosis
In a final set of experiments we studied the role of TREM-1 in the host defense against B. pseudomallei using Trem-1/3 -/mice. In contrast to the data derived from Trem-2 -/mice, no differences in bacterial counts in lung or BALF were observed between B. pseudomallei-challenged Trem-1/3 -/and WT mice (Fig 6A and 6B). In line, TREM-1 deficiency did not impact on lung pathology and cytokine levels, except for decreased KC levels, which did not influence pulmonary neutrophilic content as determined by Ly6-stainings (Fig 6E and 6F, Table 2). However, TREM-1 did influence bacterial dissemination as bacterial loads in blood and liver were significantly decreased in Trem-1/3 -/mice compared to WTs 72h after infection (P<0.01; Fig 6C and 6D). We next evaluated TREM-1's role in systemic inflammation and end organ damage. At 72h post-infection, the levels of key regulatory cytokines in the systemic compartment (TNF-α, IL-6, IL-10, MCP-1 and IFN-γ) did not differ between Trem-1/3 -/mice and WT (Table 2). Induced pathology of the spleen (Fig 6G) was similar in Trem-1/3 -/and WT mice.
In correspondence with the lower hepatic bacterial counts at 72h, we found lower levels of the hepatocellular injury markers AST and ALT levels in Trem-1/3 -/mice compared to WT mice (Fig 6H and 6I). LDH levels, reflecting general organ injury, were elevated in Trem-1/3 -/mice at 24 h, while they were reduced compared to their WT counterparts at 72h post-infection (P<0.05; Fig 6J). No difference in plasma BUN levels was observed between mice strains ( Fig 6K).   The Role of TREM-1 and -2 in Melioidosis TREM-1 deficiency does not impact on ex vivo cytokine responsiveness and phagocytosis nor intracellular killing of B. pseudomallei TREM-1 is abundantly expressed on monocytes and macrophages following exposure to B. pseudomallei [31]. In line with previous findings [11], Trem-1/3 -/-BMDM produced less TNFα in response to LPS stimulation (P<0.05; Fig 7B). Surprisingly, no differences in cellular responsiveness were found between AM and whole blood derived from WT and Trem-1/ 3 -/mice (Fig 7A-7C). Lastly, we wished to determine whether TREM-1 contributes to phagocytosis and/or killing of B. pseudomallei. No differences in phagocytic and killing capacities between WT and TREM-1 deficient BMDM were observed (Fig 7D and 7E).

Discussion
TREM-1 and TREM-2 are innate immune receptors that have demonstrated to either amplify or regulate TLR and NLR signaling after recognition of pathogen-associated molecular patterns. Our study is the first to examine the role of both TREM-1 and TREM-2 during experimental melioidosis. We observed increased TREM-1 and TREM-2 expression during experimental melioidosis, both at the local site of infection and systemically. Subsequently, we found that TREM-2 impairs the host defense against murine B.pseudomallei-induced sepsis, as demonstrated by an improved survival of infected Trem-2 -/mice as a direct result of diminished bacterial dissemination, decreased inflammation and less organ damage. Our ex vivo studies suggest that the protective effect of TREM-2 deficiency in part results from the diminished capacity of TREM-2-deficient macrophages to elicit a pro-inflammatory response which is an important contributor to organ injury in the event of sepsis. TREM-1 was also found to play a detrimental role during B. pseudomallei infection, which is in line with our earlier finding that blocking TREM-1 could improve survival during melioidosis [31]. However when compared to TREM-2 the role of TREM-1 in the host response against B. pseudomallei seems to be limited.
Previous studies have demonstrated that soluble TREM-1 levels are up-regulated in plasma of patients with sepsis, pneumonia and melioidosis [31,41,42]. In addition, it is known that surface TREM-1 expression is increased on monocytes of melioidosis patients [31]. However, soluble TREM-1 levels in septic patients do not always correlate to the expression of membrane-bound TREM-1 on different myeloid cell types [31,43]. Less is known about the kinetics of TREM-2 expression during infection. A recent study demonstrated that during sepsis TREM-2 expression on ascites-retrieved cells of patients with abdominal sepsis was increased [27]. Correspondingly, TREM-2 was up-regulated on AM of mice infected with S. pneumoniae [25]. In line with these earlier studies, we now show that both TREM-1 and TREM-2 mRNA expression is elevated in lung and liver tissue of mice infected with B. pseudomallei. Further research however is warranted to study the cell surface protein expression of TREM-2 on neutrophils and macrophages during melioidosis.
The in vivo role of TREM-2 in infectious diseases remains ill defined. In a model studying P. aeruginosa keratitis TREM-2 deficiency increased corneal bacterial loads [44]. More recently, Chen et al. demonstrated that TREM-2 is required for efficient bacterial clearance in a murine polymicrobial sepsis model using a TREM-2 blocking recombinant protein [27]. In the same study it was shown that administration of TREM-2 overexpressing bone marrow derived myeloid cells improved survival during polymicrobial sepsis, but not endotoxaemia [27]. In sharp contrast, Gawish et al. demonstrated a beneficial effect of TREM-2 deficiency during endotoxaemia [45]. The same group also observed a survival benefit of Trem-2 -/mice during S. pneumoniae pneumonia [25], while no effect on mortality of TREM-2 deficiency was seen during E. coli sepsis [45]. To evaluate how TREM-2 deficiency led to increased clearance of B. pseudomallei, we assessed the functional roles of macrophages that express TREM-2 [19,25]. TREM-2 is known to be involved in direct killing [27,44] and phagocytosis of bacteria by macrophages [24,25]. Interestingly, we did not find impaired bacterial killing or phagocytosis of B. pseudomallei by BMDM or AM of Trem-2 -/mice. Several characteristics of this facultative intracellular bacterium when compared to other bacteria might in part explain these discrepancies; B. pseudomallei is capable of invading both phagocytic and non-phagocytic cells [46] and circumvents intracellular defense mechanisms efficiently in order to replicate and spread to adjacent cells [47,48].
TREM-2 is traditionally regarded as a negative regulator of the in vitro inflammatory response in response to TLR-ligands [19,21,45] In contrast, our study now demonstrates that TREM-2 deficiency leads to a reduced inflammatory response to B. pseudomallei both ex vivo and in vivo suggesting that TREM-2's role during inflammation may not be that upfront. This is in line with recent studies investigating the role of TREM-2 in models of pneumococcal pneumonia [25], post-stroke inflammation [49] and DSS-induced colitis [50]. Different elements, can explain these inconsistencies: differences in mice strains used (BALB/C versus C57Bl/6), different experimental murine models (e.g. caecal ligation and puncture (CLP)model versus a intranasal inhalation model for sepsis), differences in TREM-2 blockade (e.g. by using TREM-2 deficient mice or TREM-2 antibodies) and lastly the difference of an in vitro approach in contrast to our ex vivo cellular challenge model. Interestingly, a recent study showed augmented inflammation by TREM-2 deficient peritoneal macrophages in response to LPS [45], while the same group observed the reversed phenotype in alveolar macrophages [25], underlining possible cell-specific functions of TREM-2. Of importance, neutrophil recruitment to the lung, an important defense mechanism during melioidosis [32,51], was reduced in Trem-2 -/mice during experimental melioidosis as determined by Ly6-staining. This may be a potential result of the decreased inflammatory response and production of chemokines following infection. In this respect, it is noteworthy, that IL-1β-which we and others have shown to be involved in excessive deleterious neutrophil influx during experimental melioidosis [37,52] -was also significantly reduced in Trem-2 -/mice. No differences were observed in the influx of macrophages (S2 Fig). Excessive inflammation and neutrophil influx and activation can lead towards multi-organ failure [53], which is almost universally seen in lethal cases of melioidosis. Distant organ injury was significantly reduced in Trem-2 -/mice, potentially as a result of a reduced influx of inflammatory cells. Trem-2 -/mice displayed an evidently reduced inflammatory response, which resulted in a strong survival benefit. In addition, it is well known that B. pseudomallei can replicate intracellularly [28], and neutrophils may act as its permissive host cell [52]. We could therefore hypothesize that the anti-inflammatory phenotype and the reduced bacterial loads seen in TREM-2 deficient mice are a result of decreased intracellular bacterial replication at the infection site, due to reduced neutrophilic influx.
Taken together, during melioidosis, TREM-2 deficiency resulted in a restricted inflammatory response, thereby decreasing organ damage and mortality. Future research should focus on the potential of anti-TREM-2 treatment of B.pseudomallei-infected mice.
TREM-1 amplifies TLR-responses and therefore might dangerously enhance the inflammatory response to bacterial infection [18]. Controversial results have been found on the role of TREM-1 during bacterial infection. TREM-1 deficiency has shown to be detrimental during endotoxaemia [17] and polymicrobial sepsis [12,54], while in contrast, moderate levels of TREM-1 can improve survival during polymicrobial sepsis, but not endotoxaemia [55]. Blockade of TREM-1 with a peptide called LP17 could partially protect mice from B. pseudomallei induced lethality [31]. In this study however, we observed, using the same infection model, that survival of B. pseudomallei-infected TREM-1-deficient mice was similar to WTs. This might be explained by the fact that these mice were completely TREM-1-deficient and in addition lacked TREM-3, a DAP12-coupled activating receptor on murine macrophages, which supposedly acts as an activating receptor [56]. In contrast, in humans TREM-3 is a pseudogene [56]. However, since DAP12 is known to both potentiate and attenuate TLR-signaling, it is perhaps not surprising that the net-effect on bacterial clearance of B. pseudomallei is not affected.
TREM-1 has other functions next to TLR-signaling enhancement, such as phagocytosis and the production of reactive oxygen species [57]. Furthermore, TREM-1 has been recently linked to trans-epithelial migration of neutrophils after infection with P. aeruginosa [15]. Blocking TREM-1 completely could therefore interfere with these important antibacterial mechanisms. We did not find a role for TREM-1 in the killing or phagocytosis of B. pseudomallei, which is in line with the fact that TREM-1/3 deficiency in neutrophils neither impacts on bacterial killing, phagocytosis and chemotaxis of P. aeruginosa [15]. This suggests that other phagocytic receptors on leukocytes are more important for the efficient eradication of B. pseudomallei [38,58,59].
Murine models like the one used here, which make use of relatively young mice exposed to an intranasal bacterial inoculum, do show inter-experiment variation, as reflected by differences in bacterial dissemination and as a result inflammation at the latter time-points before mice will succumb to infection. In addition, caution is needed when extrapolating data from murine experiments to human disease." Taking these precautions into mind, we here demonstrate that murine melioidosis is associated with increased TREM-1 and -2 expression. TREM-2 deficiency is beneficial during experimental Gram-negative sepsis caused by a clinical relevant pathogen, resulting in lower bacterial loads, reduced organ damage, decreased inflammation and improved survival. When compared to TREM-2, TREM-1 plays a limited detrimental role during experimental melioidosis. These results provide new information on the expression and function of TREM-2 during melioidosis and may demonstrate its potential therapeutic usefulness.