Role of Common γ-Chain Cytokines in Lung Interleukin-22 Regulation after Acute Exposure to Aspergillus fumigatus.

Humans are constantly exposed to the opportunistic mold Aspergillus fumigatus, and disease caused by this pathogen is often determined by the magnitude of local and systemic immune responses. We have previously shown a protective role for interleukin-22 (IL-22) after acute A. fumigatus exposure. Here, employing IL-22Cre R26ReYFP reporter mice, we identified iNKT cells, γδ T cells, and type 3 innate lymphoid cells (ILC3s) as lung cell sources of IL-22 in response to acute A. fumigatus exposure. As these cells often utilize common γ-chain cytokines for their development or maintenance, we determined the role of IL-7, IL-21, and IL-15 in lung IL-22 induction and A. fumigatus lung clearance. We observed that IL-7, IL-21, and IL-15 were essential for, partially required for, or negatively regulated the production of IL-22, respectively. Deficiency in IL-7 and IL-21, but not IL-15R, resulted in impaired fungal clearance. Surprisingly, however, the absence of IL-7, IL-21, or IL-15R signaling had no effect on neutrophil recruitment. The levels of IL-1α, an essential anti-A. fumigatus proinflammatory cytokine, were increased in the absence of IL-7 and IL-15R but decreased in the absence of IL-21. IL-7 was responsible for maintaining lung iNKT cells and γδ T cells, whereas IL-21 was responsible for maintaining lung iNKT cells and ILC3s. In contrast, IL-15R deficiency had no effect on the absolute numbers of any IL-22 cell source, rather resulting in enhanced per cell production of IL-22 by iNKT cells and γδ T cells. Collectively, these results provide insight into how the IL-22 response in the lung is shaped after acute A. fumigatus exposure.

The common γ-chain cytokine IL-7 promotes immunopathogenesis during fungal asthma.

Asthmatics sensitized to fungi are reported to have more severe asthma, yet the immunopathogenic pathways contributing to this severity have not been identified. In a pilot assessment of human asthmatics, those subjects sensitized to fungi demonstrated elevated levels of the common γ-chain cytokine IL-7 in lung lavage fluid, which negatively correlated with the lung function measurement PC20. Subsequently, we show that IL-7 administration during experimental fungal asthma worsened lung function and increased the levels of type 2 cytokines (IL-4, IL-5, IL-13), proallergic chemokines (CCL17, CCL22) and proinflammatory cytokines (IL-1α, IL-1ß). Intriguingly, IL-7 administration also increased IL-22, which we have previously reported to drive immunopathogenic responses in experimental fungal asthma. Employing IL22CreR26ReYFP reporter mice, we identified γδ T cells, iNKT cells, CD4 T cells and ILC3s as sources of IL-22 during fungal asthma; however, only iNKT cells were significantly increased after IL-7 administration. IL-7-induced immunopathogenesis required both type 2 and IL-22 responses. Blockade of IL-7Rα in vivo resulted in attenuated IL-22 production, lower CCL22 levels, decreased iNKT cell, CD4 T-cell and eosinophil recruitment, yet paradoxically increased dynamic lung resistance. Collectively, these results suggest a complex role for IL-7 signaling in allergic fungal asthma.

Acidic Mammalian Chitinase Negatively Affects Immune Responses during Acute and Chronic Aspergillus fumigatus Exposure.

Chitin is a polysaccharide that provides structure and rigidity to the cell walls of fungi and insects. Mammals possess multiple chitinases, which function to degrade chitin, thereby supporting a role for chitinases in immune defense. However, chitin degradation has been implicated in the pathogenesis of asthma. Here, we determined the impact of acidic mammalian chitinase (AMCase) (Chia) deficiency on host defense during acute exposure to the fungal pathogen Aspergillus fumigatus as well as its contribution to A. fumigatus-associated allergic asthma. We demonstrate that chitin in the fungal cell wall was detected at low levels in A. fumigatus conidia, which emerged at the highest level during hyphal transition. In response to acute A. fumigatus challenge, Chia-/- mice unexpectedly demonstrated lower A. fumigatus lung burdens at 2 days postchallenge. The lower fungal burden correlated with decreased lung interleukin-33 (IL-33) levels yet increased IL-1ß and prostaglandin E2 (PGE2) production, a phenotype that we reported previously to promote the induction of IL-17A and IL-22. During chronic A. fumigatus exposure, AMCase deficiency resulted in lower dynamic and airway lung resistance than in wild-type mice. Improved lung physiology correlated with attenuated levels of the proallergic chemokines CCL17 and CCL22. Surprisingly, examination of inflammatory responses during chronic exposure revealed attenuated IL-17A and IL-22 responses, but not type 2 responses, in the absence of AMCase. Collectively, these data suggest that AMCase functions as a negative regulator of immune responses during acute fungal exposure and is a contributor to fungal asthma severity, putatively via the induction of proinflammatory responses.

IL-33 Signaling Regulates Innate IL-17A and IL-22 Production via Suppression of Prostaglandin E2 during Lung Fungal Infection.

Members of the IL-1 family play protective and regulatory roles in immune defense against the opportunistic mold Aspergillus fumigatus In this study, we investigated the IL-1 family member IL-33 in lung defense against A. fumigatus IL-33 was detected in the naive lung, which further increased after exposure to A. fumigatus in a dectin-1-independent manner. Mice deficient in the receptor for IL-33 (Il1rl1-/-) unexpectedly demonstrated enhanced lung clearance of A. fumigatus IL-33 functioned as a negative regulator of multiple inflammatory cytokines, as IL-1α, IL-1ß, IL-6, IL-17A, and IL-22 were significantly elevated in fungal-exposed Il1rl1-/- mice. Subsequently, IL-33 administration to normal mice attenuated fungal-induced IL-17A and IL-22, but not IL-1α, IL-1ß, or IL-6, production. IL-33-mediated regulation of IL-17A and IL-22 did not involve the modulation of IL-23 but rather PGE2; PGE2 was significantly increased in fungal-exposed Il1rl1-/- mice, and normal mice produced less PGE2 after fungal exposure when administered IL-33, suggesting that IL-33-mediated regulation of IL-17A and IL-22 occurred at the level of PGE2 This was confirmed by in vivo cyclooxygenase 2 inhibition, which attenuated fungal-induced IL-17A and IL-22, as well as IL-1α, IL-1ß, and IL-6, production in Il1rl1-/- mice, resulting in impaired fungal clearance. We also show that a PGE2 receptor agonist increased, whereas a PGE2 synthase inhibitor decreased, the levels of IL-17A and IL-22 but not IL-1α, IL-1ß, or IL-6. This study establishes novel mechanisms of innate IL-17A/IL-22 production via PGE2 and regulation of the PGE2/IL-17A/IL-22 axis via IL-33 signaling during lung fungal exposure.

Eosinophil deficiency compromises lung defense against Aspergillus fumigatus.

Exposure to the mold Aspergillus fumigatus may result in allergic bronchopulmonary aspergillosis, chronic necrotizing pulmonary aspergillosis, or invasive aspergillosis (IA), depending on the host's immune status. Neutrophil deficiency is the predominant risk factor for the development of IA, the most life-threatening condition associated with A. fumigatus exposure. Here we demonstrate that in addition to neutrophils, eosinophils are an important contributor to the clearance of A. fumigatus from the lung. Acute A. fumigatus challenge in normal mice induced the recruitment of CD11b+ Siglec F+ Ly-6G(lo) Ly-6C(neg) CCR3+ eosinophils to the lungs, which was accompanied by an increase in lung Epx (eosinophil peroxidase) mRNA levels. Mice deficient in the transcription factor dblGATA1, which exhibit a selective deficiency in eosinophils, demonstrated impaired A. fumigatus clearance and evidence of germinating organisms in the lung. Higher burden correlated with lower mRNA expression of Epx (eosinophil peroxidase) and Prg2 (major basic protein) as well as lower interleukin 1ß (IL-1ß), IL-6, IL-17A, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and CXCL1 levels. However, examination of lung inflammatory cell populations failed to demonstrate defects in monocyte/macrophage, dendritic cell, or neutrophil recruitment in dblGATA1-deficient mice, suggesting that the absence of eosinophils in dlbGATA1-deficient mice was the sole cause of impaired lung clearance. We show that eosinophils generated from bone marrow have potent killing activity against A. fumigtaus in vitro, which does not require cell contact and can be recapitulated by eosinophil whole-cell lysates. Collectively, our data support a role for eosinophils in the lung response after A. fumigatus exposure.

Mesenchymal stromal cells mediate Aspergillus hyphal extract-induced allergic airway inflammation by inhibition of the Th17 signaling pathway.

Systemic administration of mesenchymal stromal cells (MSCs) suppresses airway inflammation and methacholine-induced airway hyper-responsiveness (AHR) in mouse models of T helper cell (Th) type 2-mediated eosinophilic allergic airway inflammation (AAI); however, the efficacy of MSCs in mouse models of severe Th17-mediated neutrophilic AAI has not yet been demonstrated. We assessed MSC effects in a mouse model of mixed Th2/Th17 AAI produced by mucosal exposure to Aspergillus fumigatus hyphal extract (AHE). Following sensitization produced by oropharyngeal AHE administration, systemic (tail vein) administration of syngeneic MSCs on the first day of challenge significantly reduced acute AHR predominantly through reduction of Th17-mediated airway inflammation. In parallel experiments, MSCs also mitigated AHR when administered during recurrent challenge 10 weeks after initial sensitization and challenge through reduction in systemic Th17-mediated inflammation. Investigation into potential mechanistic actions of MSCs in this model demonstrated that although T regulatory cells were increased in all AHE-treated mice, MSC administration did not alter T regulatory cell numbers in either the acute or recurrent model. Differential induction of interleukin-17a secretion was observed in ex vivo restimulation of mediastinal lymph node mixed-cell cytokine analyses. Although the mechanisms by which MSCs act to decrease inflammation and AHR in this model are not yet fully elucidated, decrease in Th17-mediated airway inflammation appears to play a significant role. These results provide a basis for further investigations of MSC administration as a potential therapeutic approach for severe refractory neutrophilic asthma.

STAT4-dependent and -independent Th2 responses correlate with protective immunity against lung infection with Pneumocystis murina.

Although it is clear that the loss of CD4(+) T cells is a predisposing factor for the development of Pneumocystis pneumonia, specific Th mechanisms mediating protection are not well understood. Th1, Th2, and Th17 responses have each been implicated in protective responses during infection. As STAT4 may promote Th1 and Th17 development, yet antagonize Th2 development, we investigated its role in Pneumocystis murina host defense. STAT4 was required for Th1 and, unexpectedly, Th2 responses in the lungs of C57BL/6 (BL/6) and BALB/c mice 14 d postchallenge, but only BALB/c Stat4(-/-) mice demonstrated susceptibility to P. murina lung infection. BL/6 Stat4(-/-), but not BALB/c Stat4(-/-), mice maintained an enhanced alternatively activated (M2) macrophage signature in the lungs, which we have previously reported to be associated with enhanced P. murina clearance. In addition, anti-P. murina class-switched Abs were increased in BL/6 Stat4(-/-) mice, but not BALB/c Stat4(-/-) mice. Supporting our experimental observations, plasma from HIV-infected individuals colonized with Pneumocystis jirovecii contained significantly lower levels of the Th2 cytokines IL-4, IL-5, and IL-13 compared with HIV-infected individuals who were not colonized. Collectively, our data suggest that robust local and systemic Th2-mediated responses are critical for immunity to Pneumocystis.

Chlorine gas exposure increases susceptibility to invasive lung fungal infection.

Chlorine (Cl2) is a highly irritating and reactive gas with potential occupational and environmental hazards. Acute exposure to Cl2 induces severe epithelial damage, airway hyperreactivity, impaired alveolar fluid clearance, and pulmonary edema in the presence of heightened inflammation and significant neutrophil accumulation in the lungs. Herein, we investigated whether Cl2 exposure affected the lung antimicrobial immune response leading to increased susceptibility to opportunistic infections. Mice exposed to Cl2 and challenged intratracheally 24 h thereafter with the opportunistic mold Aspergillus fumigatus demonstrated an >500-fold increase in A. fumigatus lung burden 72 h postchallenge compared with A. fumigatus mice exposed to room air. Cl2-exposed A. fumigatus challenged mice also demonstrated significantly higher lung resistance following methacholine challenge and increased levels of plasma proteins (albumin and IgG) in the bronchoalveolar lavage fluid. Despite enhanced recruitment of inflammatory cells to the lungs of Cl2-exposed A. fumigatus challenged mice, these cells (>60% of which were neutrophils) demonstrated a profound impairment in generating superoxide. Significantly higher A. fumigatus burden in the lungs of Cl2 exposed mice correlated with enhanced production of IL-6, TNF-α, CXCL1, CCL2, and CCL3. Surprisingly, however, Cl2-exposed A. fumigatus challenged mice had a specific impairment in the production of IL-17A and IL-22 in the lungs compared with mice exposed to room air and challenged with A. fumigatus. In summary, our results indicate that Cl2 exposure markedly impairs the antimicrobial activity and inflammatory reactivity of myeloid cells in the lung leading to increased susceptibility to opportunistic pathogens.

The ß-glucan receptor dectin-1 promotes lung immunopathology during fungal allergy via IL-22.

Sensitization to fungi, such as the mold Aspergillus fumigatus, is increasingly becoming linked with asthma severity. We have previously shown that lung responses generated via the ß-glucan receptor Dectin-1 are required for lung defense during acute, invasive A. fumigatus infection. Unexpectedly, in an allergic model of chronic lung exposure to live A. fumigatus conidia, ß-glucan recognition via Dectin-1 led to the induction of multiple proallergic (Muc5ac, Clca3, CCL17, CCL22, and IL-33) and proinflammatory (IL-1ß and CXCL1) mediators that compromised lung function. Attenuated proallergic and proinflammatory responses in the absence of Dectin-1 were not associated with changes in Ido (IDO), Il12p35/Ebi3 (IL-35), IL-10, or TGF-ß levels. Assessment of Th responses demonstrated that purified lung CD4(+) T cells produced IL-4, IL-13, IFN-γ, and IL-17A, but not IL-22, in a Dectin-1-dependent manner. In contrast, we observed robust, Dectin-1-dependent IL-22 production by unfractionated lung digest cells. Intriguingly, the absence of IL-22 alone mimicked the attenuated proallergic and proinflammatory responses observed in the absence of Dectin-1, suggesting that Dectin-1-mediated IL-22 production potentiated responses that led to decrements in lung function. To this end, neutralization of IL-22 improved lung function in normal mice. Collectively, these results indicate that the ß-glucan receptor Dectin-1 contributes to lung inflammation and immunopathology associated with persistent fungal exposure via the production of IL-22.

Administration of nitrite after chlorine gas exposure prevents lung injury: effect of administration modality.

Cl(2) gas toxicity is complex and occurs during and after exposure, leading to acute lung injury (ALI) and reactive airway syndrome (RAS). Moreover, Cl(2) exposure can occur in diverse situations encompassing mass casualty scenarios, highlighting the need for postexposure therapies that are efficacious and amenable to rapid and easy administration. In this study, we assessed the efficacy of a single dose of nitrite (1 mg/kg) to decrease ALI when administered to rats via intraperitoneal (ip) or intramuscular (im) injection 30 min after Cl(2) exposure. Exposure of rats to Cl(2) gas (400 ppm, 30 min) significantly increased ALI and caused RAS 6-24h postexposure as indexed by BAL sampling of lung surface protein and polymorphonucleocytes (PMNs) and increased airway resistance and elastance before and after methacholine challenge. Intraperitoneal nitrite decreased Cl(2)-dependent increases in BAL protein but not PMNs. In contrast im nitrite decreased BAL PMN levels without decreasing BAL protein in a xanthine oxidoreductase-dependent manner. Histological evaluation of airways 6h postexposure showed significant bronchial epithelium exfoliation and inflammatory injury in Cl(2)-exposed rats. Both ip and im nitrite improved airway histology compared to Cl(2) gas alone, but more coverage of the airway by cuboidal or columnar epithelium was observed with im compared to ip nitrite. Airways were rendered more sensitive to methacholine-induced resistance and elastance after Cl(2) gas exposure. Interestingly, im nitrite, but not ip nitrite, significantly decreased airway sensitivity to methacholine challenge. Further evaluation and comparison of im and ip therapy showed a twofold increase in circulating nitrite levels with the former, which was associated with reversal of post-Cl(2) exposure-dependent increases in circulating leukocytes. Halving the im nitrite dose resulted in no effect in PMN accumulation but significant reduction of BAL protein levels, indicating a distinct nitrite dose dependence for inhibition of Cl(2)-dependent lung permeability and inflammation. These data highlight the potential for nitrite as a postexposure therapeutic for Cl(2) gas-induced lung injury and also suggest that administration modality is a key consideration in nitrite therapeutics.

Experimental Pneumocystis lung infection promotes M2a alveolar macrophage-derived MMP12 production.

Among several bacterial and viral pathogens, the atypical fungal organism Pneumocystis jirovecii has been implicated as a contributor to the pathogenesis of chronic obstructive pulmonary disease (COPD). In a previous study, we reported that Pneumocystis-colonized HIV-positive subjects had worse obstruction of airways and higher sputum levels of macrophage elastase/matrix metalloproteinase 12 (MMP12), a protease strongly associated with the development of COPD. Here, we examined parameters of Pneumocystis-induced MMP12 in the lungs of mice and its role in the lung immune response to murine Pneumocystis. Initial studies demonstrated that P. murina exposure induced Mmp12 mRNA expression in whole lungs and alveolar macrophages (AMs), which was dependent on the presence of CD4+ T cells as well as signal transducer and activator of transcription 6. Mmp12 mRNA expression was upregulated in AMs by interleukin (IL)-4 treatment, but downregulated by interferon (IFN)-γ, indicating preferential expression in alternatively activated (M2a) macrophages. IL-4 treatment induced the 54-kDa proenzyme form of MMP12 and the 22-kDa fully processed and active form, whereas IFN-γ failed to induce either. Despite a reported antimicrobial role in macrophage phagolysosomes, mice deficient in MMP12 were not found to be more susceptible to lung infection with P. murina. Collectively, our data indicate that MMP12 induction is a component of the P. murina-induced M2 response and thus provides insight into the link between Pneumocystis colonization/infection and exacerbations in COPD.

Dietary L-arginine supplementation during gestation in mice enhances reproductive performance and Vegfr2 transcription activity in the fetoplacental unit.

Regarded as one of the most versatile amino acids, arginine serves as a precursor for many molecules and has been reported to improve the reproductive performance of rats and pigs. To this end, we sought to determine if dietary L-arginine alters fetoplacental vascular endothelial growth factor receptor-2 (Vegfr2) transcription activity. Eighteen wild-type FVB/N female mice were bred to homozygous FVB/N-Tg(Vegfr2-luc)-Xen male mice. Bred female mice received 1 of 2 experimental diets: one supplemented with 2.00% (wt:wt) L-arginine (+Arg) or 1 supplemented with 4.10% (wt:wt) alanine (+Ala) to serve as an isonitrogenous control for +Arg. In addition, 6 mice were fed a nonsupplemented control (Con) diet to normalize bioluminescent imaging data. All data were analyzed using ANOVA followed by Fisher's least significant difference. Total feed intake did not differ between groups; however, mice in the +Arg group consumed more arginine (P < 0.05). Arginine supplementation increased weight gain during the latter one-third of gestation (d 12- 18), total litter size, number of pups born alive, number of placental attachment sites, litter birth weight, and litter weight of pups born alive but decreased the individual birth weights (P < 0.05). During d 12-18, arginine supplementation increased (P < 0.05) the mean total Vegfr2 transcription activity and Vegfr2 transcription activity corrected for fetoplacental mass. Moreover, mice in the +Arg group had an earlier rise in Vegfr2 transcription activity. In conclusion, our results demonstrate that the beneficial effect of dietary L-arginine supplementation on mammalian reproduction is associated with enhanced Vegfr2 transcription activity in fetoplacental tissues.

Dectin-1-dependent interleukin-22 contributes to early innate lung defense against Aspergillus fumigatus.

We have previously reported that mice deficient in the beta-glucan receptor Dectin-1 displayed increased susceptibility to Aspergillus fumigatus lung infection in the presence of lower interleukin 23 (IL-23) and IL-17A production in the lungs and have reported a role for IL-17A in lung defense. As IL-23 is also thought to control the production of IL-22, we examined the role of Dectin-1 in IL-22 production, as well as the role of IL-22 in innate host defense against A. fumigatus. Here, we show that Dectin-1-deficient mice demonstrated significantly reduced levels of IL-22 in the lungs early after A. fumigatus challenge. Culturing cells from enzymatic lung digests ex vivo further demonstrated Dectin-1-dependent IL-22 production. IL-22 production was additionally found to be independent of IL-1ß, IL-6, or IL-18 but required IL-23. The addition of recombinant IL-23 augmented IL-22 production in wild-type (WT) lung cells and rescued IL-22 production by lung cells from Dectin-1-deficient mice. In vivo neutralization of IL-22 in the lungs of WT mice resulted in impaired A. fumigatus lung clearance. Moreover, mice deficient in IL-22 also demonstrated a higher lung fungal burden after A. fumigatus challenge in the presence of impaired IL-1α, tumor necrosis factor alpha (TNF-α), CCL3/MIP-1α, and CCL4/MIP-1ß production and lower neutrophil recruitment, yet intact IL-17A production. We further show that lung lavage fluid collected from both A. fumigatus-challenged Dectin-1-deficient and IL-22-deficient mice had compromised anti-fungal activity against A. fumigatus in vitro. Although lipocalin 2 production was observed to be Dectin-1 and IL-22 dependent, lipocalin 2-deficient mice did not demonstrate impaired A. fumigatus clearance. Moreover, lung S100a8, S100a9, and Reg3g mRNA expression was not lower in either Dectin-1-deficient or IL-22-deficient mice. Collectively, our results indicate that early innate lung defense against A. fumigatus is mediated by Dectin-1-dependent IL-22 production.

Neutrophils produce interleukin 17A (IL-17A) in a dectin-1- and IL-23-dependent manner during invasive fungal infection.

We have previously reported that compromised interleukin 17A (IL-17A) production in the lungs increased susceptibility to infection with the invasive fungal pathogen Aspergillus fumigatus. Here we have shown that culturing lung cells from A. fumigatus-challenged mice ex vivo demonstrated Dectin-1-dependent IL-17A production. In this system, neutralization of IL-23 but not IL-6, IL-1ß, or IL-18 resulted in attenuated IL-17A production. Il23 mRNA expression was found to be lower in lung cells from A. fumigatus-challenged Dectin-1-deficient mice, whereas bone marrow-derived dendritic cells from Dectin-1-deficient mice failed to produce IL-23 in response to A. fumigatus in vitro. Addition of recombinant IL-23 augmented IL-17A production by wild-type (WT) and Dectin-1-deficient lung cells, although the addition of IL-6 or IL-1ß did not augment the effect of IL-23. Intracellular cytokine staining of lung cells revealed lower levels of CD11b(+) IL-17A(+) and Ly-6G(+) IL-17A(+) cells in A. fumigatus-challenged Dectin-1-deficient mice. Ly-6G(+) neutrophils purified from the lungs of A. fumigatus-challenged Dectin-1-deficient mice displayed lower Il17a mRNA expression but surprisingly had intact Rorc and Rora mRNA expression. We further demonstrated that Ly-6G(+) neutrophils required the presence of myeloid cells for IL-17A production. Finally, upon in vitro stimulation with A. fumigatus, thioglycolate-elicited peritoneal neutrophils were positive for intracellular IL-17A expression and produced IL-17A in a Dectin-1- and IL-23-dependent manner. In summary, Dectin-1-dependent IL-17A production in the lungs during invasive fungal infection is mediated in part by CD11b(+) Ly-6G(+) neutrophils in an IL-23-dependent manner.

In vivo monitoring of fetoplacental Vegfr2 gene activity in a murine pregnancy model using a Vegfr2-luc reporter gene and bioluminescent imaging.

BACKGROUND: Vascular endothelial growth factor receptor-2 (VEGFR2) plays a pivotal role in angiogenesis by eliciting vascular endothelial cell growth when bound to VEGF, a powerful pro-angiogenic ligand. While Vegf and Vegfr2 are expressed throughout gestation, the latter third of gestation in mice is characterized by a marked increase in fetoplacental angiogenesis. Thus, the objective of this study was to determine the feasibility of monitoring fetoplacental Vegfr2 gene activity non-invasively using a Vegfr2-luc reporter transgenic mouse and bioluminescent imaging. METHODS: Imaging parameters were optimized using two wild-type (WT) females, bearing Vegfr2-luc fetuses. Then, seven WT females, bred to Vegfr2-luc males, were imaged from gestational day (GD) 12 to 18 to determine the usefulness of the Vegfr2-luc mouse as a model for studying fetoplacental Vegfr2 activity during pregnancy. Semi-quantitative RT-PCR of Vegfr2 was also performed on whole fetoplacental units during this time. Additionally, resultant neonates were imaged at postnatal day (PND) 7, 14 and 21 to monitor Vegfr2 activity during post-natal development. RESULTS: Fetoplacental Vegfr2 gene activity was detected as light emissions beginning on GD 12 of gestation and increased throughout the imaging period (P < 0.05), and this paralleled the Vegfr2 mRNA data obtained from RT-PCR analysis. A decline in fetoplacental light emissions was associated with a poor pregnancy outcome in one pregnancy, indicating that this approach has potential use for studies monitoring pregnancy well being. Additionally, neonatal Vegfr2 activity was detected at PND 7, 14 and 21 but declined with time (P < 0.0001). CONCLUSIONS: In utero fetoplacental Vegfr2 gene activity was monitored longitudinally in a quantitative manner using a luciferase reporter gene and bioluminescent imaging during the latter third of gestation. This study demonstrates the feasibility of using the Vegfr2-luc mouse to monitor late gestation fetoplacental angiogenic activity under normal and experimental conditions. Additionally, neonatal Vegfr2 gene activity was monitored for three weeks postpartum, allowing continuous monitoring of Vegfr2 activity during the latter third of gestation and postnatal development within the same animals.