Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in premature infants (1, 2). While the initial determinants of survival in patients with NEC reflect the extent of intestinal disease, long-term outcomes are attributable to the extent of disease in other organs, especially the lung (3, 4). In particular, NEC-induced lung injury is more severe and more difficult to treat than the bronchopulmonary dysplasia that affects many premature infants, and represents a major complication of NEC. While estimates vary, NEC-induced lung injury develops in approximately 10% to 15% of infants with NEC (5), yet the underlying molecular processes leading to this lung damage remain incompletely understood.
In seeking to address the gaps in our understanding of the mechanisms of NEC-associated lung disease, we recently determined that the development of NEC requires the activation of the lipopolysaccharide receptor, toll-like receptor 4 (TLR4) on the intestinal epithelium, which leads to induction of pro-inflammatory Th17 cells and a reduction in anti-inflammatory T regulatory lymphocytes (Tregs) (1, 6, 7). The subsequent release of the endogenous TLR4 agonist high mobility group box-1 (HMGB1) from the damaged intestinal epithelium activates TLR4 on the clara-positive cells of the pulmonary epithelium, resulting in neutrophil influx and damage to the premature lung (8). Importantly, the full extent of the cellular events that lead to the induction of NEC-induced lung injury, and in particular the role, if any, of the adaptive immune system in its pathogenesis remains unknown.
We now hypothesize that the development of NEC-induced lung injury requires a TLR4-mediated increase in pro-inflammatory Th17 cells and a reduction in Tregs in the lung, and that strategies to reduce Th17 cells and increase Tregs can reduce the extent of NEC induced lung injury. In support of this hypothesis, we now show that NEC-induced lung injury is associated with an induction of Th17 cells in the lungs of mice and humans with NEC that was required for induction of the lung disease as the adoptive transfer of CD4+ T cells isolated from lungs of mice with NEC into the lungs of immune incompetent mice (Rag1−/− mice) induced profound inflammation in the lung, while the depletion of Tregs exacerbated NEC induced lung injury. Blocking the receptor for the Th17 cell-specific pro-inflammatory cytokine IL-17 or the Th17 cell recruiting chemokine CCL25 prevented inflammation in mouse lung with NEC, while the aerosolized delivery of all trans-retinoic acid (ATRA) to boost Tregs reduced lung inflammation. Strikingly, the instillation of mouse lung with the novel TLR4 small molecule inhibitor compound 34 (C34) or deletion of TLR4 from the Surfactant protein C-1 (Sftpc1) positive cells in the lungs restored the Treg/Th17 balance and reduced the degree of NEC-induced lung injury. These findings reveal that gut-lung signaling, through pulmonary epithelial TLR4, is required for the induction of NEC-induced lung injury through alterations of lymphocyte populations in the newborn lung, and indicate that reversal of Treg/Th17 imbalance can serve as a novel approach for the reduction of this devastating complication of NEC.
MATERIALS AND METHODS
Reagents and antibodies
Sources of antibodies and other reagents were as follows: cleaved caspase-3 (Cell Signaling, Danver, Mass), DAPI (Invitrogen, Waltham, Mass), inducible nitric oxide synthase (iNOS, BD Bioscience, San Jose, Calif), Brdu (Fischer Scientific, Waltham, Mass). The novel TLR4 inhibitor Compound 34 (2-acetamidopyranoside, C17H27NO9, MW 389) was described by our group recently, and synthesized as in our published reports (9).
The animal experiments described in these studies were approved by the Johns Hopkins University Animal Care Committee (Protocol Number: M014M362) and were performed according the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. C57BL/6, Sftpctm1(cre/ERT2)Blh, Rag1–/– (RagB6.129S7-Rag1tm1Mom/J), IL-17-GFP (B6.129P2[Cg]-Rorctm2Litt/J), B6.129-Foxp3tm3(DTR/GFP)Ayr/J (Foxp3+DTR) mice were obtained from the Jackson Laboratory (Farmington, Conn) and housed in an specific pathogen-free facility.
To generate a mouse line in which TLR4 gene was specifically excised from type II penumocytes (TLR4Δsftpc1), Tlr4loxP mice were cross-bred with Sftpctm1(cre/ERT2)Blh mice (Jackson Labs). The progeny was found to lack TLR4 in the type II penumocytes as determined by PCR, and to lack an inflammatory response to the intra-tracheal instillation of LPS (Supplemental Figure 1, http://links.lww.com/SHK/A812).
Human lung samples
Human infant lung samples were obtained and processed at autopsy from patients with NEC or age-matched controls, with approval from the University of Pittsburgh Institutional Review Board (CORID No. 491) and in accordance with the University of Pittsburgh and Johns Hopkins University anatomical tissue procurement guidelines. All samples were de-identified via an independent honest broker assurance mechanism (Approval #: HB#043) and transferred to Johns Hopkins University under the guidance of MTA approval (JUH MTA # A26558) for analysis.
Experimental necrotizing enterocolitis in mice
Experimental necrotizing enterocolitis was induced in 6 to 8-day-old mice as we have reported and validated in previous studies (6, 7, 10). In brief, neonatal mouse pups were gavage fed formula [Similac Advance infant formula (Abbott Nutrition):Esbilac (PetAg) canine milk replacer, 2:1] that was supplemented with enteric bacteria obtained from an infant with necrotizing enterocolitis requiring surgery, five times per day and exposed to hypoxia (5% O2, 95% N2) for 10 min in a hypoxic chamber (Billups-Rothenberg) twice daily for 4 days. This protocol induces the development of patchy necrosis and cytokine induction that mimics that seen in human NEC (11). For tracheal administration of Atra, recombinant IL-10, and Compound 34 (all 10 mg/kg), the reagents were administered via inhalation 1 day before the initiation of the NEC model and then one dose every day for the duration of the model.
Quantitative real-time PCR, immunohistochemistry
Quantitative real-time PCR was performed using the Bio-Rad CFX96 real-time system as described previously (6), using the primers listed in Table 1. Total RNA was isolated from samples of either lung or the terminal ileum of mice, or specimens resected from human infants during surgery. The expression levels of the pro-inflammatory cytokines were measured relative to the housekeeping gene RPLO. Immunofluorescent staining was performed on 4% paraformaldehyde-fixed 5 μM-thick paraffin sections, following antigen retrieval was employed as described (6), and assessed on a Zeiss LSM710 confocal microscope.
Flow cytometry for IL-17A+ and Foxp3+ cells
Flow cytometry was performed in single-cell suspensions from mouse lung as reported (12) before and in brief as follows: freshly harvested sections of mouse lung were minced and incubated in 50 μg/mL Liberase solution (Roche, Indianapolis, Ind) for 30 min at 37°C and agitated at 750 rpm. The cells were then disassociated with an 18-gauge needle. The tissue digest was passed through a 40 μm cell strainer into a tube with wash buffer and centrifuged at 400 × g, 4°C for 5 min. The pellet was then resuspended in 50 mL 1% BSA (VWR Life Sciences, Bridgeport, NJ) in PBS and centrifuged at 400 × g, 4°C for 5 min and the supernatant was discarded. The cell pellet was resuspended at 2.5 × 107 cells/mL in FACS buffer. Single-cell suspensions were then incubated with anti-CD16/CD32 (BD Bioscience, San Jose, Calif) to block Fc receptor binding (20 min, 4°C). IL-17A+ and Foxp3+ cells were analyzed according to manufacturer's guidance on a BD Accuri6 flow cytometer.
Immunofluorescent staining of lung tissues was performed on 4% paraformaldehyde-fixed 5 μM-thick paraffin sections. The sections were first warmed to 56°C in a vacuum incubator (Isotemp Vacuum Oven, Fisher Scientific) then washed immediately in xylene, gradually redehydrated in ethanol (100%, 95%, 70%, water), and then processed for antigen retrieval in citrate buffer (10 mM pH6.0)/microwave (1,000 watt, 6 min). Samples were then washed with PBS, blocked with 1% BSA/5% donkey-serum (1 h, room temperature), then incubated overnight at 4°C with primary antibodies (1:200 dilutions in 0.5% BSA), washed three times with PBS, incubated with appropriate fluorescent labeled secondary antibodies (1:1,000 dilution in 0.5% BSA, Life Technologies Inc) and the nuclear marker DAPI (Invitrogen, Waltham, Mass). Slides were then mounted using Gelvatol (Sigma-Aldrich, Allentown, Pa) solution prior to imaging using a Zeiss LSM 710 Confocal microscope (Carl Zeiss, Jena, Germany) under appropriate filter sets. Antibodies used in mouse lung immunostainings are: cleaved caspase 3 (Biocare Medical, rabbit anti rat/mouse), DAPI (Life Sciences) iNOS (mouse anti rat/mouse, BD Bioscience, San Jose, Calif) and Brdu (Fischer Scientific, rat).
Isolation of CD4+ T cells from neonatal lung and adoptive transfer to Rag1−/− mice
The neonatal lung lobes were mechanically homogenized with a razor blade and then digested in DMEM medium containing 10% fetal bovine serum, 50 μM 2-mercaptoethanol, 250 U/mL collagenase IV and 25 U/mL DNase I as previously (13). After debris removal using 70 μm cell strainers, the total lung lymphoid cells were isolated using a 40% and 60% Percoll gradient, and the lung CD4+ T cells were further purified using CD4 MicroBeads (Miltenyl Biotec, Calif) according to the manufacturer's protocol, and 1×105 isolated CD4+ T cells were instilled into Rag1−/− mouse lung through tracheal intubation. Mice were euthanized and evaluated for histology and cytokine expression after 24 h.
CCL25 and IL-17 ELISA
Sandwich ELISA analysis was performed according to the manufacturer's instructions (mouse CCL25 and IL-17 Duoset). Briefly, capture antibody was incubated on 96-well flat-bottomed plates overnight. Plates were washed and blocked with 5%BSA (1 h, room temperature) and samples were added to the plate, incubated overnight (4oC), washed extensively then incubated with biotinylated detection antibody (2 h, room temperature). Following washes with 0.05% tween-20 in PBS, streptavidin alkaline phosphatase was added to the wells and the enzymatic reaction was stopped after 30 min by the addition of an equal volume of 0.2N sulfuric acid and the color change was read on a spectrophotometer (450 nm, Molecular Dynamics). Data were normalized to the standards according to manufacturer's instructions and quantified using Graph Pad (Prism).
Data were analyzed for statistical significance by two-tailed Student t test or analysis of variance (ANOVA) using Prism 7 software (GraphPad). Statistical significance was determined as having a P value of less than 0.05 and data are represented as mean ± SEM as indicated. All experiments were repeated at least in triplicate, with at least six pups per group.
NEC is associated with increased Th17 cells and reduced Tregs in the developing lungs of mice
The development of NEC in mice and humans was associated with significant lung injury (Fig. 1), consistent with our prior observations (8), and revealed by histologic evidence of alveolar destruction and cellular infiltration of the lungs (Fig. 1, A and B) and the pro-inflammatory molecule iNOS is detected by immunostaining (Fig. 1, A and B). Importantly, the presence of NEC-induced lung injury in humans was characterized by an increase in Th17 cells as transcriptional factor ROR as revealed by immunostaining and RT-PCR (Fig. 1, A and G), and by a reduction in Tregs, as revealed by reduced expression of the transcription factor Foxp3 by immunohistochemistry (Fig. 1A) and RT-PCR (Fig. 1H). The imbalance of Th17/Treg in the lung in NEC was also observed in a well-validated mouse model of the disease, which involves subjecting newborn mice to 4 days of formula gavage, hypoxia, and administration of stool from an infant with severe NEC (6, 10). As indicated in Figure 1B, I, and J, mice expressing GFP downstream of the IL-17 promoter (i.e., IL-17 GFP+ mice) that were subjected to experimental NEC showed significantly more GFP+ (i.e., IL-17) cells in the lung as compared with control mice (Fig. 1, B and I), suggesting that NEC induces the influx of Th17 cells into the lung. By contrast, we observed significantly fewer Foxp3+ cells in the lungs of mice expressing GFP downstream of the Foxp3 promoter (i.e., Foxp3 GFP+ mice) with NEC (Fig. 1, B and J). Taken together, these results show an imbalance of Th17/Treg in the lungs of humans and mice with NEC characterized by an increase in Th17 cells and a reduction in Tregs.
An induction of Th17 cells and reduction in Tregs is required for the development of NEC-induced lung injury
To determine whether the increase in Th17 cells and reduction in Tregs observed in the lungs of mice and humans with NEC is required for the induction of NEC induced lung injury, we undertook three parallel approaches. First, we administered inhibitory antibodies to IL-17 directly into the lung via inhalation, which significantly reduced the extent of NEC-induced lung injury compared with mice receiving non-specific IgG, as revealed by histology and immunostaining for iNOS (Fig. 2A), and by qRT-PCR for expression of the pro-inflammatory genes iNOS and IL-6 (Fig. 2, B and C). Next, we removed Tregs from Foxp3iDTR mice by administration of diphtheria toxin daily throughout the model, which resulted in significantly more severe NEC-induced lung injury, as manifest by more severe disruption of the lung histology (Fig. 2D), and more severe upregulation of iNOS and IL-6 (Fig. 2, E and F). In a final set of experiment, we assessed the effects of adoptively transferring the CD4+ T cells from the lungs of mice with NEC into Rag1−/− mice on the development of lung injury. As shown in Figure 1B, I, J, cells harvested from the lungs of mice with NEC were found to be predominantly Th17 cells (Fig. 1, B, I, J), and their adoptive transfer into the airway of Rag1−/− mice that lack T or B cells resulted in spontaneous lung injury after 24 h, as manifest by histologic destruction of the airways (Fig. 2G), and upregulation of iNOS and TNF (Fig. 2, H and I) in the lungs as compared with Rag1−/− mice receiving cells from uninjured controls. These findings reveal that the T cell milieu in the inflamed lung plays a causative role in the development of NEC-induced lung injury. We therefore next sought to understand the mechanisms that mediated the Treg/Th17 lymphocyte imbalance in the lung, and then evaluated whether reversal of the lymphocyte imbalance could attenuate the degree of NEC-induced lung injury.
TLR4 activation on Sftpc1 cells is required for altering the Th17/Treg balance in the development of NEC-induced lung injury in mice
We next sought to evaluate the proximal signaling mechanisms that mediate the Th17/Treg imbalance that we have observed to be critical in the development of NEC-induced lung injury. In our previous studies, we demonstrated that TLR4 activation on Clara positive lung epithelial cells is critical in the development of NEC-associated lung injury (8), suggesting the possibility that TLR4 signaling in the lung could similarly impact Th17/Treg composition. Moreover, the expression of TLR4 in the lungs of mice increased in the postnatal period and peaked at the time points corresponding to the beginning of the NEC model, i.e., days p5–p10 (Fig. 3A). To test directly whether TLR4 signaling on the lung could influence the development of NEC-induced lung injury, we first generated mice that selectively lack TLR4 on the type 2 pneumocytes (Sftpc1-positive pulmonary cells) as described in Methods. The progeny, herein called TLR4Δsftpc1 mice, were healthy and fertile, displayed no obvious lung phenotype, reproduced at expected Mendelian ratios, and did not significantly induce pro-inflammatory cytokines in response to intratracheal LPS as compared with wild-type mice, confirming the success of the deletion strategy (Supplemental Figure 1, http://links.lww.com/SHK/A812). Importantly, when subjected to the model of experimental NEC, TLR4Δsftpc1 mice still developed significant intestinal injury (Fig. 3, B–D), yet the previously observed imbalance in the Treg/Th17 populations was reversed by inhibition of TLR4 on the Sftpc1 cells (Fig. 3, E, H, I). Furthermore, TLR4Δ sftpc1 mice showed significantly reduced histological evidence of lung injury (Fig. 3E), and decreased expression of the pro-inflammatory molecules iNOS and IL-6 (Fig. 3, F and G), consistent with the observation that inhibition of TLR4 on the pulmonary epithelium reduces NEC-induced lung injury.
In further support of the role of TLR4 signaling in the development of NEC-induced lung injury, we administered by aerosolizing our recently discovered TLR4 inhibitor compound 34 that has the molecular formula of C17H27NO9, an oligosaccharide with the molecular weight of 389.40, also known as C34 (9, 14). The administration of C34 prevented the increase in Th17 cells and reduced the influx of Tregs in the lung, and attenuated the degree of NEC-induced lung injury (Fig. 3, E–I). It is noteworthy that the aerosolized administration of C34 did not reduce the degree of intestinal injury in mice with NEC (Fig. 3, A–C), consistent with pulmonary delivery, as we reported previously (8). Taken in aggregate, these findings illustrate that TLR4 signaling on the sftp1 cells leads to an imbalance in Th17 and Tregs in the lung, causing NEC-induced lung injury. We therefore next sought to determine the upstream mechanisms involved.
TLR4 induction of CCL25 in the mouse lung leads to Th17 infiltration and NEC-induced lung injury
To understand the mechanisms by which TLR4 signaling on the pulmonary epithelium leads to NEC-induced lung injury, we next focused on the expression of chemokine (C–C motif) ligand 25 (CCL25), which is a chemokine for the recruitment of pro-inflammatory lymphocytes into the lung (15, 16). As shown in Figure 4, the expression of CCL25 in wild-type mice was significantly increased in the lungs after induction of NEC, as revealed by RT-PCR and ELISA (Fig. 4, A and B). Importantly, inhibition of CCL25 by administration of neutralizing antibody, reduced the degree of NEC-induced lung injury (Fig. 4F) and restored the Treg/Th17 imbalance (Fig. 4, C and D), consistent with its role in the recruitment of Th17 cells in the lung. Importantly, the induction of CCL25 was lost in TLR4Δ sftpc1 mice exposed to experimental NEC (Fig. 4, H and I), confirming that TLR4 signaling in the Sftpc1 cells is required for CCL25 induction, which leads to the lymphocyte imbalance in the lung.
Increasing the population of Tregs in the newborn lung reduces NEC-induced lung injury
In the final series of studies, we evaluated whether increasing the population of Tregs in the lung could attenuate NEC-induced lung injury and provide a window for intervention in the setting of NEC. To do so, we undertook two approaches: first, we administered all trans-retinoic acid (Atra) to induce Tregs through enhancing Foxp3 expression and inhibiting Th17 polarization (17–19); second, we administered the Treg cytokine IL-10, both before and during NEC model, to mimic the function of Tregs in the lung. As shown in Figure 5, the administration of Atra restored Foxp3+ Tregs in the lungs of mice exposed to experimental NEC to levels similar to that of control mice as expected (Fig. 5, A and B), while also significantly reducing the extent of NEC-induced lung injury as revealed by improved histology (Fig. 5C) and reduced expression of iNOS and IL-6 in the lung (Fig. 5, D and E). Further, the aerosolized administration of IL-10, that is released from Tregs, significantly reduced the extent of NEC-induced lung injury (Fig. 5, C–E). These findings reveal not only the functional importance of the Th17/Treg imbalance in the pathogenesis of NEC-induced lung injury, but also show that restoration of the Treg/Th17 imbalance can open a therapeutic window of opportunity to limit the degree of NEC-induced lung injury.
In the current study, we reveal that the induction of NEC-induced lung injury reflects the imbalance between anti-inflammatory Tregs and pro-inflammatory Th17 cells in the lung, and that development of this imbalance requires TLR4 signaling in the Sftpc1-positive pulmonary cells. We further show that strategies to increase Treg function or distribution, or to limit Th17 function, can reduce the extent of NEC-induced lung injury. The reversibility of the process is a critical finding in the overriding goal of advancing our ability to care for children with NEC-induced lung injury.
Previously, our group has developed a mouse model of NEC in which lung injury develops that shares important features with the human condition (8). Using this model, we demonstrated a critical role for TLR4 signaling on the Clara cells of the pulmonary epithelium in the development of NEC-associated lung disease through recruiting pro-inflammatory neutrophils into the lung (8). We now extend these prior findings by focusing on the adaptive immune system, and the observation that a relative imbalance exists between Th17 and Treg cells in the lungs of mice and humans with NEC. We also confirmed that the imbalance is a cause rather than a consequence of NEC-induced lung inflammation, as the adoptive transfer of the lymphocyte population of the injured lung induces disease in a naive Rag1−/− mouse. Although a causative role for Th17 cells in NEC-induced lung injury is now apparent, it is not clear what the exact function of the Th17 cells is in causing lung injury, nor whether there may be an interaction between Th17 cells and the neutrophils in the development of lung injury. In this regard, previous authors have revealed that enhanced influx of Th17 cells exacerbated lung inflammation by secreting pro-inflammatory cytokine and chemokine and facilitating inflammatory neutrophil infiltration in the lung in various disease settings (20–23), suggesting the possibility that similar interactions may also underlie disease in the lungs of infants with NEC.
The current work sheds light on the mechanisms that lead to the induction of Th17 cells in the lung of neonatal mice and humans. In naive conditions, Th17 cells are rarely present in the lung, especially in the neonate (22, 24, 25), although recent studies have shown that their recruitment into the lung is mediated in part by CCL25 (15, 16, 26). We now reveal a TLR4-dependent induction of CCL25 in the lungs of mice with NEC, and further reveal that CCL25 is induced in NEC as a mediator of the lymphocyte influx (Fig. 4). We acknowledge that controversy exists regarding the role of CCL25 in recruiting lymphocytes into the lung, and in fact others have shown that CCL2 and CCL20 might also contribute to increased Th17 influx into the lung in a variety of disease settings (27, 28). A role for these other chemokines in NEC-induced lung injury, and other factors that could enhance Th17 cell populations in the lung in the setting of lung injury including differentiation from naive T cells, remain to be examined.
The current findings are consistent with prior reports of the protective roles for Tregs in lung injury models including bacterial pneumonia, trauma-induced lung injury, and irradiation-induced lung injury (29–33). It is uncertain what the TLR4 ligand may be that induces Tregs on the Sftpc1 cells in the current study, yet given our prior work, HMGB1 release may play a role, as may gut-derived bacterial products. The current study illustrates several potential translational findings that could lead to new approaches for limiting the development of NEC-induced lung injury. For instance, the administration of a TLR4 inhibitor compound 34 (C34) directly into the airway had a protective role in NEC-induced lung injury by interrupting the pathways leading to injurious Th17 enrichment and restoring Treg/Th17 homeostasis; furthermore, aerosol delivery of Atra into mouse lung in experimental NEC model led to restoration of the shifted balance of Th17/Treg and protection of NEC-induced lung injury. These findings have clinical potential, given that current therapies for lung injury in the premature babies are largely insufficient (34, 35). These potential benefits are balanced by limitations in the current study. For instance, the findings may be applicable uniquely to the neonates, before epithelial and immune cells reach full maturity. In addition, our current findings, especially those related to mechanistic details, are only validated in our mouse model, and the relevance to other models of lung injury, and indeed other animal models of NEC such as experimental swine and rat models is unknown. Further analysis of human tissue and the assessment of these mechanistic roles in large animal models of NEC would be fruitful next steps. Moreover, toll-like receptors other than TLR4, such as toll-like receptor 2 (TLR2) that displayed an enhanced gene expression in mouse lung underwent NEC (Supplemental Figure 2, http://links.lww.com/SHK/A813), might also play roles in NEC-induced lung injury and need to be further investigated.
Taken together, our current findings illustrate a pivotal role of TLR4 signaling on Sftpc1 cells in causing an induction of Th17 cells, a loss of Tregs, and the development of NEC-induced lung injury. These studies also reveal that manipulating the activity of TLR4 signaling and the balance of Th17/Treg in the lung may prevent and treat this devastating complication of NEC.
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