Transepithelial migration of neutrophils into the lung requires TREM-1

Acute respiratory infections are responsible for more than 4 million deaths each year. Neutrophils play an essential role in the innate immune response to lung infection. These cells have an armamentarium of pattern recognition molecules and antimicrobial agents that identify and eliminate pathogens. In the setting of infection, neutrophil triggering receptor expressed on myeloid cells 1 (TREM-1) amplifies inflammatory signaling. Here we demonstrate for the first time that TREM-1 also plays an important role in transepithelial migration of neutrophils into the airspace. We developed a TREM-1/3–deficient mouse model of pneumonia and found that absence of TREM-1/3 markedly increased mortality following Pseudomonas aeruginosa challenge. Unexpectedly, TREM-1/3 deficiency resulted in increased local and systemic cytokine production. TREM-1/3–deficient neutrophils demonstrated intact bacterial killing, phagocytosis, and chemotaxis; however, histologic examination of TREM-1/3–deficient lungs revealed decreased neutrophil infiltration of the airways. TREM-1/3–deficient neutrophils effectively migrated across primary endothelial cell monolayers but failed to migrate across primary airway epithelia grown at the air-liquid interface. These data define a new function for TREM-1 in neutrophil migration across airway epithelial cells and suggest that it amplifies inflammation through targeted neutrophil migration into the lung.

Acute respiratory infections have been described as the forgotten pandemic, responsible for 4.5 million deaths annually. Effective neutrophil migration into the intrapulmonary airspace is essential for pathogen elimination in the lung. Neutrophils possess diverse tools, including ROS intermediates, antimicrobial peptides, neutrophil extracellular traps, and numerous pattern recognition receptors (PRRs), that facilitate the response to de novo infections (1, 2). Moreover, neutrophils participate in B cell maturation and differentiation, Th17 responses, dendritic cell maturation, natural killer cell responses, and IL-10 production, revealing a regulatory role for neutrophils (2). Like other cells of the innate immune system, neutrophils express several large genetically encoded receptor families that act to detect and eliminate pathogens. These families include the TLR family, C-type lectin receptors, and the triggering receptor expressed on myeloid cells (TREM) family (3–5).

TREM-1, which was initially discovered on human neutrophils and monocytes, has been studied in patients with pneumonia and sepsis (6, 7). Increased TREM-1 levels in bronchoalveolar lavage (BAL) fluid strongly correlate with ventilator-associated pneumonia, and systemic levels of TREM-1 in the serum serve as a marker of sepsis (8, 9). TREM-1 has been shown to amplify the inflammatory cascade in response to infection (10). Surface expression of TREM-1 is increased in response to LPS and other microbial products in vitro. Antibody-mediated crosslinking of TREM-1 induces modest cellular activation and proinflammatory cytokine secretion. Furthermore, when TREM-1 acts in concert with PRRs, there is a synergistic increase in inflammatory signaling (10–13). Such data suggest that TREM-1 functions as an amplifier of the immune response in the context of microbial infection.

During the process of pathogen recognition and elimination, excessive cytokine and chemokine release can lead to extensive tissue damage and even death (14, 15). Therapeutic agents designed to temper the deleterious consequences of host-pathogen interactions result in a survival benefit in some experimental models of endotoxemia. On the other hand, it is clear that some inflammation is necessary for bacterial killing in vivo. Accordingly, numerous clinical examples of impaired neutrophil function are linked to increased rates of infection and death, including chronic granulomatous disease (CGD), neutropenic fever, and lymphocyte adhesion deficiency. However, animal studies indicate that effective bacterial clearance can be achieved in the setting of decreased inflammation (16, 17). In the case of TREM-1, blockade of TREM-1 has been achieved by administering soluble forms of TREM-1, small molecule blocker (LP-17), or RNA silencing (10, 18, 19). All three of these interventions result in decreased systemic cytokine production and improved survival in mice. Taken together, these animal studies of TREM-1 blockade suggest that it may be possible to modulate the TREM-1 pathway to decrease inflammation without sacrificing bacterial control.

We hypothesized that TREM-1 deficiency would decrease inflammation and improve survival in a murine pneumonia model. Paradoxically, we found that TREM-1/3 deficiency resulted in a marked increase in mortality. Our data suggest that impaired neutrophil transepithelial migration is the mechanism underlying the failure to clear lung bacteria, resulting in increased mortality with TREM-1/3 deficiency. These findings identify a novel role for TREM-1 in neutrophil epithelial transmigration in addition to its established role in amplifying inflammatory signaling.

Generation of TREM-1–deficient mice. The goal of these studies was to elucidate the role of TREM-1 in pneumonia using a mouse model of TREM-1 deficiency. In mice, the Trem1 gene is adjacent to a highly homologous gene, Trem3, which likely arose from a duplication event. The two receptors have similar cellular distribution and associate with DAP12. Further suggesting that these two proteins have redundant functions in the mouse, functional studies in our laboratory and others show that both molecules are amplifiers of inflammatory signaling (20). In contrast, Trem3 is a pseudogene in humans, and therefore no functional overlap exists. Thus, to model the effect of blocking TREM-1 in humans, we generated a TREM-1/3–deficient mouse. To abrogate expression of functional TREM-1 and TREM-3 proteins, we made a targeting construct in which an 8.8-kb fragment containing most of exon 3 and exon 4 of Trem1 as well as exon 1 of Trem3 was replaced with a MC1neopA gene flanked by loxP sites (Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI64181DS1). This effectively deleted the transmembrane region of TREM-1 in addition to the start site and first domain of TREM-3. Chimeras generated from a correctly targeted clone were bred to transgenic mice expressing Cre under the CMV promoter on a C57BL/6 background to delete the MC1neopA gene. Homozygote TREM-1/3–deficient mice failed to express TREM-1 and TREM-3 proteins (Supplemental Figure 2). Inheritance of this mutation followed expected Mendelian distribution, with 25% homozygotes, 21% WT, and 54% heterozygotes assayed (n = 229). Flow cytometric analysis using lineage markers revealed no significant differences in the percentages of circulating leukocyte populations as a result of Trem1 and Trem3 deletion (Supplemental Figure 3).

TREM-1/3 is essential for host survival following pulmonary challenge with P. aeruginosa. To examine the role of TREM-1 in survival in response to pneumonia, we challenged TREM-1/3–deficient mice, WT littermate controls, or WT C57BL/6 mice intratracheally with P. aeruginosa. Survival analysis performed using Gehan-Breslow-Wilcoxon statistical tests showed a significant increase in mortality in TREM-1/3–deficient mice as compared with WT mice, with χ² = 18.75 and P < 0.0001 (Figure 1A). Additional clinical indicators of sepsis including body temperature and evidence of end-organ damage were monitored. TREM-1/3–deficient animals were hypothermic (Figure 1B, P = 0.0001), consistent with the development of systemic disease. TREM-1/3–deficient animals also showed evidence of end-organ damage in the form of transaminitis as measured by serum alanine aminotransferase (ALT) levels, whereas WT animals had no elevation (Figure 1C). These data indicate that TREM-1/3 expression is essential for host survival following pulmonary challenge with P. aeruginosa. Challenge with heat-killed P. aeruginosa resulted in no mortality in WT or TREM-1/3–deficient mice (n = 4 in each group, data not shown), suggesting that the difference in survival observed in the TREM-1/3–deficient mice was due to a decrease in the ability to control active infection and not increased inflammation in the setting of endotoxin challenge.

Figure 1

TREM-1/3 expression is essential for host survival following P. aeruginosa challenge. (A) Kaplan-Meier survival plot of TREM-1/3–deficient (n = 35, 4 experiments) and WT mice (n = 26, 4 independent experiments) following intratracheal challenge with P. aeruginosa (*P = 0.0001). (B) Body temperature was measured 14 hours after infection with P. aeruginosa (*P = 0.0001) (n = 95 mice, 9 experiments). (C) As a marker of liver damage, ALT levels were measured 14 hours after intratracheal challenge with P. aeruginosa (n = 11).

TREM-1/3–deficient animals fail to eradicate P. aeruginosa. To assess bacterial clearance, we performed bacterial sampling of BAL fluid using whole lung lavage on anesthetized animals at either 4 or 14 hours after infection. At 4 hours after infection, both TREM-1/3–deficient and WT BAL fluid (Figure 2A) showed growth of P. aeruginosa. However, at 14 hours after infection, P. aeruginosa CFU were higher in the lungs of TREM-1/3–deficient animals (Figure 2B, P = 0.011). In addition, P. aeruginosa CFU were also significantly higher in cultures obtained from whole lung suspensions generated 14 hours after infection in TREM-1/3–deficient as compared with WT mice (Figure 2C, P = 0.036). Next, blood was obtained by cardiac puncture 14 hours after infection to assess systemic bacteremia (Figure 2D). TREM-1/3–deficient animals had a higher incidence of bacteremia (53% TREM-1/3–deficient vs. 27% WT). Thus, TREM-1/3–deficient mice fail to eradicate P. aeruginosa infection locally, resulting in systemic dissemination of bacteria and death.

Figure 2

TREM-1/3–deficient mice fail to eradicate P. aeruginosa following pulmonary challenge. (A) CFU of P. aeruginosa in BAL fluid at 4 hours after infection in WT and TREM-1/3–deficient mice (n = 12, 1 experiment). (B) CFU of P. aeruginosa in BAL from WT and TREM-1/3–deficient mice 14 hours after infection (*P = 0.011) (n = 27, 2 independent experiments). (C) CFU of P. aeruginosa recovered in whole lung homogenates from WT and TREM-1/3–deficient mice 14 hours after infection (*P = 0.036) (n = 14, 1 experiment). (D) CFU of P. aeruginosa recovered from cardiac puncture 14 hours after inoculation in TREM-1/3–deficient and WT mice (n = 27, 2 independent experiments).

TREM-1/3–deficient neutrophils kill P. aeruginosa effectively in vitro. To assess whether impaired neutrophil function was the mechanism for poor local and systemic control of P. aeruginosa in the TREM-1/3–deficient animals, we measured phagocytosis and in vitro neutrophil killing of P. aeruginosa. First, phagocytosis was measured using Texas red–zymosan. Neutrophils were isolated from TREM-1/3–deficient and WT bone marrow (pooled 3–5 per group). There were no significant differences in the percentage of neutrophils that ingested particles (Figure 3A) or in the number of particles phagocytized per neutrophil as determined by the geometric mean of Ly6G-positive cells (Figure 3B). To distinguish between intracellular and extracellular particles, we performed fluorescence quenching with trypan blue (21). No significant difference in quenching was detected between WT and TREM-1/3–deficient neutrophils (Supplemental Figure 4). Next, to examine the capacity of murine neutrophils to kill P. aeruginosa, we isolated neutrophils and co-incubated them with bacteria (22). At 30 minutes, more than 90% of the bacteria were ingested. Non-ingested bacteria were killed with gentamicin, as previously described (23). Intracellular bacterial killing was assessed at 30, 60, and 90 minutes after exposure to gentamicin. There was no significant difference in the ability of TREM-1/3–deficient and WT neutrophils to kill P. aeruginosa (Figure 3C). Finally, to determine how TREM-1/3 deficiency affects neutrophil ROS production, we isolated neutrophils from WT and TREM-1/3–deficient animals and stimulated them with opsonized zymosan and PMA. In the presence of no agonist, neither set of neutrophils produced ROS (Supplemental Figure 5), indicating that the neutrophils had not been activated in the isolation process. However, TREM-1/3–deficient neutrophils had a 50% reduction in ROS production in response to opsonized zymosan as compared with WT neutrophils (Supplemental Figure 5), whereas ROS production in response to PMA was intact (Supplemental Figure 5). It is well established that neutrophils eliminate P. aeruginosa through a number of O₂-independent pathways, including bactericidal/permeability-increasing protein (BPI), proteases, lipases, and defensins (24). In fact, neutrophils isolated from patients with CGD (an inherited defect in NADPH oxidase resulting in severely impaired ROS production) effectively kill P. aeruginosa, indicating that ROS production is not required in order to effectively kill this organism (24). Therefore, given the in vitro killing data, the striking in vivo phenotype in the TREM-1/3–deficient mice is likely not due to an intrinsic neutrophil killing defect.

Figure 3

TREM-1/3–deficient neutrophils effectively kill P. aeruginosa. (A) TREM-1/3–deficient and WT neutrophil phagocytosis of Texas red–labeled zymosan particles was quantitated as the percentage of cells ingesting particles (2 independent experiments; pooled neutrophils from 2–3 mice per group). (B) The relative number of Texas red–labeled particles ingested per neutrophil was also quantified using the geometric MF of Texas red–positive TREM-1/3–deficient and WT neutrophils. (C) The ability of TREM-1/3–deficient and WT bone marrow neutrophils to kill P. aeruginosa was measured (3 independent experiments; pooled neutrophils from 2–3 mice per group).

Lungs from TREM-1/3–deficient mice have decreased histologic evidence of pneumonia. Histologic examination of lungs harvested from live mice at 14 hours after infection showed an unexpected difference. Lung tissue from 5 TREM-1/3–deficient animals and 5 WT animals was analyzed by a veterinary pathologist unaware of sample identities. The degree of inflammation was quantified as described in the Methods. TREM-1/3–deficient animals had less evidence of inflammation/pneumonia as compared with WT animals (Figure 4, A and B). Moreover, TREM-1/3–deficient animals had reduced leukocyte infiltration by morphometry (P = 0.016) and fewer necropurulent foci (P = 0.05) present in the lungs as compared with WT animals (Figure 4, C and D, respectively). Taken together, these data indicate that, despite increased bacterial load and higher mortality, TREM-1/3–deficient animals have impaired pulmonary inflammation in response to P. aeruginosa challenge.

Figure 4

TREM-1/3–deficient mice have decreased histologic evidence of pneumonia following P. aeruginosa challenge. (A and B) Representative H&E-stained lungs from (A) WT or (B) TREM-1/3–deficient mice 14 hours after infection with P. aeruginosa. Original magnification, ×10; inset, ×60. Arrows indicate areas of necropurulent foci. ×10 scale bar: 140 μm; ×60 scale bar: 50 μm. (C) Leukocyte infiltration score in lungs from WT and TREM-1/3–deficient mice 14 hours after infection (n = 10 mice, *P = 0.016). (D) Necrotic foci score in lungs of WT and TREM-1/3–deficient mice 14 hours after infection (n = 10 mice, *P = 0.05). Error bars indicate SEM.

TREM-1/3–deficient animals have decreased neutrophils in the airway. To assess leukocyte migration into the lung, first we asked whether there was a defect in wbc translocation into the lung. Single-cell suspension of lungs from WT and TREM-1/3–deficient animals were prepared to quantitate total lung wbc. TREM-1/3–deficient animals had similar numbers of eosinophils, macrophages, NK cells, CD4⁺ T cells, CD8⁺ T cells, and B cells (Figure 5A, n = 15). Unexpectedly, the number of total lung neutrophils was significantly higher in TREM-1/3–deficient animals (Figure 5A, P = 0.001). Therefore, despite having more total lung neutrophils, TREM-1/3–deficient mice failed to develop pneumonia with consolidation and were unable to clear P. aeruginosa lung infection, resulting in a marked increase in mortality.

Figure 5

Quantification of neutrophils in lung compartments following P. aeruginosa challenge. (A) Quantification of total wbc populations in single-cell suspensions from lungs of WT and TREM-1/3–deficient mice 14 hours after infection (n = 15, *P = 0.001). (B) Quantification of neutrophils present in BAL of WT and TREM-1/3–deficient mice 14 hours after infection (n = 20, *P = 0.004, 2 independent experiments) (C) Morphometric analysis of the neutrophils in the interstitium of terminal bronchioles from the lungs of WT and TREM-1/3–deficient mice (n = 14, *P = 0.001). (D) Stereotactic analysis of alveolar neutrophils from the lungs of WT and TREM-1/3–deficient animals (n = 14, P = 0.79). Error bars indicate SEM.

Next, we determined using BAL whether TREM-1/3–deficient mice have defective neutrophil translocation into the airspace. More than 96% of all cells from the BAL fluid of both groups were Ly6G positive and CD115 negative (data not shown), consistent with murine neutrophil identity. TREM-1/3–deficient mice had a 50% reduction in the number of airway neutrophils (Figure 5B, P = 0.004), suggesting impaired migration of neutrophils into the airspace. We then used a stereological approach to further localize neutrophils in the compartments of the lung by specifically examining the interstitium, the airway lumen (terminal bronchioles), and the alveolar space. To assess the location of neutrophils in the distal airways, we identified terminal bronchioles and a sequestration index was calculated to assess the relative number of neutrophils in the interstitium versus the distal airway lumen. Terminal bronchioles from TREM-1/3–deficient mice had a higher sequestration index as compared with WT mice (Figure 5C, n = 13, P = 0.001), indicating that a higher proportion of neutrophils was present in the interstitial space as compared with the lumen and suggesting a neutrophil migratory block into the bronchiolar airway. There was no detectable difference in the number of neutrophils in the alveolar compartment in WT and TREM-1/3–deficient mice (Figure 5D). The features of consolidation and alveolar collapse were at times confounding issues, and it remains unclear whether a migratory block at the alveolar level exists in TREM-1/3–deficient mice. Taken together, the histologic data and BAL data indicate that, with TREM-1/3 deficiency, there is a defect in neutrophil migration into the airspace.

TREM-1/3–deficient mice have increased cytokine production both locally and systemically following P. aeruginosa challenge. Because TREM-1 acts synergistically with TLR and other PRRs to augment proinflammatory cytokine production, we hypothesized that a blunted cytokine response to infection with P. aeruginosa would be responsible for decreased airway neutrophils in TREM-1/3–deficient animals. Cytokines were sampled in the plasma, BAL fluid, and total lung homogenates. To avoid survivor bias, all samples were collected at 14 hours following infection, when all animals in both groups were alive. First, to ascertain whether decreased neutrophil migration was the result of a deficiency in local chemokine production, we obtained BAL fluid from infected mice. CXCL1/KC, CXCL2/MIP-2, and CCL2/MCP were elevated in TREM-1/3–deficient as compared with WT mice, consistent with sufficient local production of chemoattractants (Figure 6, BAL: P = 0.048, P = 0.017, and P = 0.004, respectively). In separate experiments, total tissue cytokine levels were measured in lungs harvested from anesthetized TREM-1/3–deficient and WT mice after perfusion with PBS from the right ventricle to remove intravascular blood (Figure 6, Lung homogenates). TREM-1/3–deficient mice had higher levels of IL-1α (P = 0.038), IL-1β (P = 0.038), IL-6 (P = 0.015), IL-10 (P = 0.05), IL-17 (P = 0.022), and TNF-α (P = 0.007) and chemokines CXCL1/KC (P = 0.02), CXCL2/MIP-2 (P = 0.012), and CCL2/MCP-1 (P = 0.035). Based on these results and those shown in Figures 4 and 5, we conclude that despite elevated local cytokines and chemokines, TREM-1/3–deficient animals have ineffective accumulation of neutrophils in the airway lumen.

Figure 6

TREM-1/3–deficient animals have intact local cytokine and chemokine production. BAL: Indicated chemokine levels were measured in BAL fluid from WT and TREM-1/3–deficient mice 14 hours after infection. *CXCL1/KC: P = 0.048, CCL2/MCP: P = 0.017, and CXCL2/MIP-2: P = 0.004 (n = 27, 3 independent experiments). Lung homogenates: Cytokine and chemokine profiles from WT and TREM-1/3–deficient lung homogenates. *IL-1α: P = 0.038, IL-1β: P = 0.038, IL-6: P = 0.015, IL-10: P = 0.05, IL-17: P = 0.022, TNF-α: P = 0.007, CXCL1/KC: P = 0.02, CXCL2/MIP-2: P = 0.012, and CCL2/MCP-1: P = 0.035 (n = 22, 2 independent experiments). Plasma: Plasma cytokine and chemokine levels in WT and TREM-1/3–deficient mice at 14 hours after inoculation with P. aeruginosa. *G-CSF: P = 0.0004, IL-6: P = 0.0001, IL-1α: P = 0.0448, CCL5/RANTES: P = 0.022, TNF-α: P = 0.0072, IL-10: P = 0.0007, CXCL1/KC: P = 0.0011, CXCL2/MIP-2: P = 0.0018, and CCL2/MCP-1: P = 0.0002 (n = 28, 3 independent experiments). Error bars indicate SEM.

Additionally, systemic cytokine levels were measured (Figure 6). The TREM-1/3–deficient mice had statistically significant increases in the proinflammatory cytokines G-CSF, IL-6, IL-1α, CCL5/RANTES, TNF-α, IL-12p40 (P = 0.004, data not shown), and IL12-p70 (P = 0.020, data not shown); increased levels of chemoattractants CXCL1 /KC, CCL2/MCP-1, CXCL2/MIP-2; and increased anti-inflammatory cytokine IL-10. These data indicate that the cytokine production was not impaired in the setting of infection despite the loss of the TREM-1/3 amplifying pathway. Moreover, these data indicate that impaired cytokine production was not responsible for the decrease in neutrophils in the airway lumen of TREM-1/3–deficient animals.

TREM-1/3–deficient neutrophils have normal cell death. To investigate whether the diminished number of neutrophils in the airways of TREM-1/3–deficient animals was due to increased cell death, we isolated neutrophils from TREM-1/3–deficient and WT animals and determined cell death by propidium iodide uptake as previously described (25). We observed no significant difference in the percentage of neutrophils undergoing cell death spontaneously or in response to LPS or IFN-γ in the absence or presence of serum (Figure 7, A and B, respectively). Therefore, impaired neutrophil survival is likely not the mechanism of decreased neutrophil accumulation in the airways of TREM-1/3–deficient animals.

Figure 7

TREM-1/3–deficient neutrophils undergo cell death at rates similar to those of WT neutrophils. (A and B) WT and TREM-1/3–deficient neutrophil cell death was measured in vitro at rest (NS, not stimulated) and with stimulation with LPS or IFN-γ in the absence (A) or presence (B) of FCS. Representative data, 3 independent experiments. Error bars indicate SEM.

In vitro chemokine-induced migration is intact in TREM-1/3–deficient neutrophils. Next, we examined neutrophil migration using the TX migration plate (see Methods) to determine whether the defect in neutrophil accumulation in the airway of TREM-1/3–deficient mice was the result of an intrinsic defect in the ability of TREM-1/3–deficient neutrophils to respond to chemoattractants. Neutrophils were placed in the upper compartment of chambers separated from the lower compartment by a filter with 3-μm pores. Zymosan-activated serum (ZAS) or CXCL-1/KC was added to the lower chamber as a chemoattractant. There was no significant difference in migration between WT and TREM-1/3–deficient cells (Figure 8), indicating that deficiency in TREM-1/3 does not alter the migratory response to chemoattractants.

Figure 8

Deficiency of TREM-1/3 does not abrogate in vitro neutrophil chemotaxis in response to chemokines. WT and TREM-1/3–deficient neutrophil chemotaxis was measured in response to chemoattractants KC (5 nM, 50 nM) and 10% ZAS. The line labeled PBS indicates the number of chemotactic neutrophils relative to the PBS control. Representative data; n = 2–3 mice pooled per group; 3 independent experiments. Error bars indicate SEM.

TREM-1/3–deficient neutrophils have intact transendothelial migration. The first step in neutrophil migration into infected airways is neutrophil migration from the vasculature across the pulmonary capillary endothelial cell monolayer. We hypothesized that TREM-1/3 may interact with endothelial cell receptors to facilitate neutrophil migration across the endothelial cell barrier. Therefore, we examined whether TREM-1/3–deficient neutrophils had an impaired ability to cross endothelial cell monolayers in vitro. Murine endothelial cells lines (SVEC4-10 and murine brain endothelial cells [MBECs]) were seeded on 3-μm-pore-size Transwell filters and grown to confluence. Freshly isolated bone marrow–derived neutrophils were loaded on the apical side of the endothelial monolayers, and ZAS (10%), a neutrophil chemoattractant, was added to the opposite side. There was no defect in the ability of TREM-1/3–deficient neutrophils to migrate across murine endothelial cell monolayers as compared with WT neutrophils (Figure 9). In addition, we repeated the experiments using primary murine endothelial cells cultured as previously described (26). Consistent with the cell line data, we observed similar migration of TREM-1/3–deficient and WT neutrophils across primary murine endothelial cell monolayers (Figure 9). These data suggest that impaired transendothelial migration is not the mechanism of decreased neutrophils in the airway of TREM-1/3–deficient mice following P. aeruginosa challenge.

Figure 9

TREM-1/3–deficient neutrophils have intact transendothelial migration. The ability of WT and TREM-1/3–deficient neutrophils to migrate across murine endothelial cell monolayers (SVEC4-10 cells and MBECs) and primary murine lung endothelial cell monolayers was measured in response to stimulation with 10% ZAS. Representative data, n = 2–3 mice pooled per group, 3 independent experiments. Error bars indicate SEM.

TREM-1/3–deficient neutrophils have a defect in transepithelial migration. In vivo, once neutrophils migrate from the capillaries, they must cross a pseudo-stratified epithelium to enter the luminal side of the airway. Airway epithelial cells (AECs) are polarized, and neutrophil transepithelial migration occurs from the basolateral side of AEC to the apical side of AEC into the airway lumen. Therefore, the ability of TREM-1/3–deficient neutrophils to migrate across primary polarized AECs was examined. While TREM-1 and TREM-3 are not known to be expressed on AECs, to eliminate an AEC defect in TREM-1/3–deficient mice as the source of impaired migration, we cultured both WT and TREM-1/3–deficient primary murine tracheal AECs on Transwells using previously described methods (27). The differentiated AECs from WT and TREM-1/3–deficient mice established transepithelial resistance greater than 500 Ω•cm², indicating the presence of tight junctions. Transmission electron micrograph (TEM) images of the murine airway epithelial monolayers demonstrate ciliate cell features (Figure 10A). Figure 10B depicts a neutrophil attached to underside of the Transwell filter before migration. Freshly isolated bone marrow–derived neutrophils (pooled from 3 mice per group) were loaded on the basolateral side of AECs by inversion of the Transwells. CXCL1/KC was added as a chemoattractant to the culture medium on the apical side of the epithelium. Migration of WT neutrophils was similar across WT and TREM-1/3–deficient AECs (P = 0.19). By contrast, TREM-1/3–deficient neutrophils demonstrated a significant migratory defect in response to CXCL1/KC across both WT (Figure 10C) and TREM-1/3–deficient AECs (Figure 10D). Treatment of WT neutrophils with a TREM-1 polyclonal antibody but not a TREM-3 monoclonal antibody decreased migration across WT AECs (data not shown). Together, these data suggest that TREM-1 functions in neutrophil migration across AECs and that impaired neutrophil migration is the defect responsible for the susceptibility of TREM-1/3–deficient mice to P. aeruginosa pneumonia.

Figure 10

TREM-1/3 are required for neutrophil migration across AEC monolayers. (A) TEM image the air-interface cultured primary murine airway epithelia, including 2 ciliated cells (C). (B) TEM image of a neutrophil (N) attached to the underside of the Transwell filter prior to migration through a pore. Brackets indicate the filter; arrowheads indicate each side of a pore. Scale bars: 2 μm. (C) Migration of WT and TREM-1/3–deficient neutrophils across WT AECs in response to KC or PBS control was measured. *P = 0.02 vs. WT neutrophil migration to KC. (D) WT and TREM-1/3–deficient neutrophil migration across TREM-1/3–deficient AECs was measured as in C. *P = 0.02 vs. WT neutrophil migration to KC. Representative data; n = 3 mice pooled per group; 2 independent experiments. Error bars indicate SEM.

More than 2 million people die of pneumonia annually worldwide, most of them children under the age of 5 (28). These deaths occur at extremes of the immune response: too much inflammation and its associated lung injury can cause death, and insufficient inflammation and failure to clear infection can lead to the same result. Here, we identify TREM-1 as a novel receptor required for neutrophil transepithelial migration into the airway lumen. Our data suggest a mechanism by which impaired migration results in decreased neutrophil recruitment and ineffective bacterial clearance. Subsequently, the TREM-1–deficient animals die due to excessive cytokine release and bacterial dissemination. Thus, potential therapeutic agents to treat pneumonia, such as TREM-1 inhibitors, must achieve a balance between pathogen control and host tissue damage.

The neutrophil response is titrated not only through regulated cytokine and antimicrobial product release but also via controlled migration into the lung. Indeed, recent studies using intravital microscopy document the complexity of neutrophil migration (29, 30). These cells can immediately cross an endothelial cell barrier, pause and decide not to cross, or even cross and then reverse course to return to the vessel lumen, suggesting that the neutrophil is capable of integrating complex environmental signals (30). Determining how neutrophil migration is influenced by these disparate signals is critical for understanding the pathogenesis of pneumonia, acute respiratory distress syndrome, and other inflammatory diseases of the lung. The role of TREM-1 in cytokine augmentation has been well described, and our data demonstrate that TREM-1 is also involved in the process of neutrophil transepithelial migration.

Neutrophil migration into infected tissues has been studied extensively (31, 32). In the systemic circulation, the initial site of emigration is the postcapillary venule. Here, the neutrophil undergoes capture and rolling on the endothelial surface, and selectin family members play a critical role (33–35). Once firm adhesion has been established, transendothelial migration occurs under these laminar flow conditions. In contrast, neutrophil migration in the lung occurs in the pulmonary capillaries. Here a complex web of anastomosing capillaries requires the neutrophil to deform in order to pass through the vessels (36–38). Spatial constraints in the capillary most likely supplant the need for rolling and capture in the pulmonary circulation (39), thus providing an explanation for the fundamental difference in neutrophil migration in the lung compared with the systemic circulation. On the pulmonary endothelium, firm adhesion is primarily mediated by the interaction between the β₂ integrins CD18/CD11a (also LFA-1 or αLβ2) and CD18/CD11b (also Mac-1 or αMβ2) expressed on the neutrophils and ICAM-1 (also CD54), ICAM-2, and ICAM-3 expressed on the endothelial cell surface (40–42). Both homotypic and heterotypic interactions between PECAM-1 (CD31), CD177, β₂ integrins, ICAM-1, and junctional adhesion molecule (JAM) family members facilitate transendothelial migration (43–46). Because we observed decreased neutrophil accumulation in the lung airspace in TREM-1/3–deficient mice, we initially hypothesized that TREM-1/3 deficiency would cause defective transendothelial migration. However, TREM-1/3–deficient neutrophils migrated effectively across both murine endothelial cell lines and primary murine endothelial cells, likely via these previously described pathways, suggesting that TREM-1/3 is not required for transendothelial migration.

Compared with transendothelial migration, the molecular details of transepithelial migration are less well understood, including the extent to which neutrophils infiltrate the lung at the level of the large airway, the small airway, and/or the alveolar epithelia. Moreover, transepithelial migration is a dynamic process, and neutrophil passage through the alveolar interstitium is difficult to quantitate macroscopically. Our in vitro data using large airway epithelia grown at the air-liquid interface support a defect in neutrophil transepithelial migration. However, a limitation of our study is that we are unable to culture alveolar epithelia at the air-liquid interface for in vitro testing. In our model of TREM-1/3 deficiency, we detected differential compartmentalization of neutrophils in the interstitial space at the level of the small airway in vivo, consistent with a defect in transepithelial migration.

The integrity of the airway epithelium is achieved via tight junctions and adherens junction protein interactions. Paracellular migration is presumably achieved through alterations in tight junction protein interactions. Neutrophil adherence to the pulmonary epithelial cell layer is mediated by β₂ integrins, in particular CD18/CD11b and ICAM, though CD11a and CD11c may play some role (47–55). The role of JAMs in intestinal transepithelial migration has also been studied. For example, JAM C but not JAM A has been implicated in intestinal neutrophil transepithelial migration, with studies demonstrating that JAM C antibodies decrease the rate of neutrophil transepithelial migration but do not block migration (56). Recently a role for junctional adhesion molecule–like protein (JAML), which is expressed on neutrophils, was described in intestinal transepithelial migration (57). JAML binds to epithelial cell via the coxsackie and adenovirus receptor (CAR), and soluble forms of either of these molecules impede neutrophil transepithelial migration across cultured intestinal epithelium. CD47, which is expressed on both epithelial cells and neutrophils, is also required for intestinal neutrophil transepithelial migration, though the mechanism is still being characterized and no studies have been conducted in the lung epithelium (58). To date there is no evidence for an interaction between TREM-1 and any of these adhesion molecules, suggesting that alternative mechanisms exist by which TREM-1 mediates neutrophil adhesion and migration across epithelial cells.

In this study, we demonstrate that loss of TREM-1/3 expression results in impaired neutrophil transepithelial migration in a murine pneumonia model. Gibot and colleagues examined the role of TREM-1 in bacterial pneumonia using a different approach (17). Their study involved instillation of a TREM-1–blocking peptide, LP-17, into the rat trachea following pneumonia challenge. Interestingly, this resulted in decreased lung inflammation and improved survival. Blockade of TREM-1–mediated amplification of cytokine production was thought to be the mechanism behind improved survival. However, our data suggest that LP-17 may play a role in blocking neutrophil migration. We propose that a partial blockade of neutrophil migration into the airways may allow a sufficient quantity of neutrophils to clear the infection but prevent extensive lung injury by attenuating transepithelial migration.

In summary, our results implicate TREM-1 as a novel player in neutrophil pulmonary transepithelial migration. We hypothesize that the TREM-1 interaction with the pulmonary epithelium provides a second novel regulatory pathway in vivo for TREM-1 to amplify inflammation through targeted migration into lung. In the absence of TREM-1, in vivo neutrophil migration to the airway lumen is impaired. This leads to ineffective bacterial killing and increased mortality. Further study of the role of TREM-1/3 molecules in transepithelial migration may identify therapeutic targets to modulate neutrophil influx, thereby altering the local inflammatory response not through classic cytokine signaling but through controlled migratory access.

View Supplemental data

We thank Bill Nauseef, Beverly Davidson, and Kristina Thiel for critical reading and editing of the manuscript. We also thank Mark Schultz for assistance with the MBECs. This work was supported by grants from the American Heart Association to J. Klesney-Tait (0675028N) and from the NIH to M. Colonna (GM077279-04). All imaging was carried out by the Central Microscopy Research Facilities. The data presented herein were obtained at the Flow Cytometry Facility, which is a Carver College of Medicine Core Research Facilities/Holden Comprehensive Cancer Center Core Laboratory at the University of Iowa. The Facility is funded through user fees and the financial support of the Carver College of Medicine, Holden Comprehensive Cancer Center, and Iowa City Veteran’s Administration Medical Center.

Address correspondence to: Julia Klesney-Tait, Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, 200 Hawkins Dr., Iowa City, Iowa 52242, USA. Phone: 319.356.3576; Fax: 319.353.6406; E-mail: julia-klesney-tait@uiowa.edu.

Conflict of interest: The authors have declared that no conflict of interests exists.

Reference information: J Clin Invest. 2013; 123(1):138–149. doi:10.1172/JCI64181.

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