COX-2 expression and function in the hyperalgesic response to paw inflammation in mice

Peripheral inflammation and edema are often accompanied by primary and secondary hyperalgesia which are mediated by both peripheral and central mechanisms. The role of cyclooxygenase-2 (COX-2)-mediated prostanoid production in hyperalgesia is a topic of substantial current interest. We have established a murine foot-pad inflammation model in which both pharmacologic and genetic tools can be used to characterize the role of COX-2 in hyperalgesia. Zymosan, an extract from yeast, injected into the plantar surface of the hindpaw induces an edema response and an increase in COX-2 expression in the hindpaw, spinal cord and brain. Zymosan-induced primary hyperalgesia, measured as a decrease in hindpaw withdrawal latency in response to a thermal stimulus, is long-lasting and is not inhibited by pre-treatment with the systemic COX-2 selective inhibitor, parecoxib (20 mg/kg). In contrast, the central component of hyperalgesia, measured as a reduction in tail flick latency in response to heat, is reduced by parecoxib. Zymosan-induced primary hyperalgesia in Cox-2-/- mice is similar to that of their Cox-2+/+ littermate controls. However, the central component of hyperalgesia is substantially reduced in Cox-2-/- versus Cox-2+/+ mice, and returns to baseline values much more rapidly. Thus pharmacological data suggest, and genetic experiments confirm, (i) that primary hyperalgesia in response to zymosan inflammation in the mouse paw is not mediated by COX-2 function and (ii) that COX-2 function plays a major role in the central component of hyperalgesia in this model of inflammation.     

Different forms of glycine- and GABAA-receptor mediated inhibitory synaptic transmission in mouse superficial and deep dorsal horn neurons

Background

Inflammation-mediated hyperalgesia involves tissue acidosis and sensitization of nociceptors. Many studies have reported increased expression of acid-sensing ion channel 3 (ASIC3) in inflammation and enhanced ASIC3 channel activity with pro-inflammatory mediators. However, the role of ASIC3 in inflammation remains inconclusive because of conflicting results generated from studies of ASIC3 knockout (ASIC3 ^-/-) or dominant-negative mutant mice, which have shown normal, decreased or increased hyperalgesia during inflammation.

Results

Here, we tested whether ASIC3 plays an important role in inflammation of subcutaneous tissue of paw and muscle in ASIC3 ^-/- mice induced by complete Freund's adjuvant (CFA) or carrageenan by investigating behavioral and pathological responses, as well as the expression profile of ion channels. Compared with the ASIC3 ^+/+ controls, ASIC3 ^-/- mice showed normal thermal and mechanical hyperalgesia with acute (4-h) intraplantar CFA- or carrageenan-induced inflammation, but the hyperalgesic effects in the sub-acute phase (1–2 days) were milder in all paradigms except for thermal hyperalgesia with CFA-induced inflammation. Interestingly, carrageenan-induced primary hyperalgesia was accompanied by an ASIC3-dependent Nav1.9 up-regulation and increase of tetrodotoxin (TTX)-resistant sodium currents. CFA-inflamed muscle did not evoke hyperalgesia in ASIC3 ^-/- or ASIC3 ^+/+ mice, whereas carrageenan-induced inflammation in muscle abolished mechanical hyperalgesia in ASIC3 ^-/- mice, as previously described. However, ASIC3 ^-/- mice showed attenuated pathological features such as less CFA-induced granulomas and milder carrageenan-evoked vasculitis as compared with ASIC3 ^+/+ mice.

Conclusion

We provide a novel finding that ASIC3 participates in the maintenance of sub-acute-phase primary hyperalgesia in subcutaneous inflammation and mediates the process of granuloma formation and vasculitis in intramuscular inflammation.     

TRPA1 receptor stimulation by hydrogen peroxide is critical to trigger hyperalgesia and inflammation in a model of acute gout

Acute gout attacks produce severe joint pain and inflammation associated with monosodium urate (MSU) crystals leading to oxidative stress production. The transient potential receptor ankyrin 1 (TRPA1) is expressed by a subpopulation of peptidergic nociceptors and, via its activation by endogenous reactive oxygen species, including hydrogen peroxide (H₂O₂), contributes to pain and neurogenic inflammation. The aim of this study was to investigate the role of TRPA1 in hyperalgesia and inflammation in a model of acute gout attack in rodents. Inflammatory parameters and mechanical hyperalgesia were measured in male Wistar rats and in wild-type (Trpa1^+/+) or TRPA1-deficient (Trpa1^−/−) male mice. Animals received intra-articular (ia, ankle) injection of MSU. The role of TRPA1 was assessed by receptor antagonism, gene deletion or expression, sensory fiber defunctionalization, and calcitonin gene-related peptide (CGRP) release. We found that nociceptor defunctionalization, TRPA1 antagonist treatment (via ia or oral administration), and Trpa1 gene ablation abated hyperalgesia and inflammatory responses (edema, H₂O₂ generation, interleukin-1β release, and neutrophil infiltration) induced by ia MSU injection. In addition, we showed that MSU evoked generation of H₂O₂ in synovial tissue, which stimulated TRPA1 producing CGRP release and plasma protein extravasation. The MSU-elicited responses were also reduced by the H₂O₂-detoxifying enzyme catalase and the reducing agent dithiothreitol. TRPA1 activation by MSU challenge-generated H₂O₂ mediates the entire inflammatory response in an acute gout attack rodent model, thus strengthening the role of the TRPA1 receptor and H₂O₂ production as potential targets for treatment of acute gout attacks.     

Transcriptional responses of neonatal mouse lung to hyperoxia by Nrf2 status

Hyperoxia exposure can inhibit alveolar growth in the neonatal lung through induction of p21/p53 pathways and is a risk factor for the development of bronchopulmonary dysplasia (BPD) in preterm infants. We previously found that activation of nuclear factor erythroid 2 p45-related factor (Nrf2) improved survival in neonatal mice exposed to hyperoxia likely due to increased expression of anti-oxidant response genes. It is not known however, whether hyperoxic induced Nrf2 activation attenuates the growth impairment caused by hyperoxia in neonatal lung. To determine if Nrf2 activation modulates cell cycle regulatory pathway genes associated with growth arrest we examined the gene expression in the lungs of Nrf2^−/− and Nrf2^+/+ neonatal mice at one and 3 days of hyperoxia exposure.MethodsMicroarray analysis was performed in neonatal Nrf2^+/+ and Nrf2^−/− lungs exposed to one and 3 days of hyperoxia. Sulforaphane, an inducer of Nrf2 was given to timed pregnant mice to determine if in utero exposure attenuated p21 and IL-6 gene expression in wildtype neonatal mice exposed to hyperoxia.ResultsCell cycle regulatory genes were induced in Nrf2^−/− lung at 1 day of hyperoxia. At 3 days of hyperoxia, induction of cell cycle regulatory genes was similar in Nrf2^+/+ and Nrf2^−/− lungs, despite higher inflammatory gene expression in Nrf2^−/− lung.Conclusionp21/p53 pathways gene expression was not attenuated by Nrf2 activation in neonatal lung. In utero SUL did not attenuate p21 expression in wildtype neonatal lung exposed to hyperoxia. These findings suggest that although Nrf2 activation induces expression of anti-oxidant genes, it does not attenuate alveolar growth arrest caused by exposure to hyperoxia.     

Collagen receptors integrin alpha2beta1 and discoidin domain receptor 1 regulate maturation of the glomerular basement membrane and loss of integrin alpha2beta1 delays kidney fibrosis in COL4A3 knockout mice

Maturation of the glomerular basement membrane (GBM) is essential for maintaining the integrity of the renal filtration barrier. Impaired maturation causes proteinuria and renal fibrosis in the type IV collagen disease Alport syndrome. This study evaluates the role of collagen receptors in maturation of the GBM, matrix accumulation and renal fibrosis by using mice deficient for discoidin domain receptor 1 (DDR1), integrin subunit α2 (ITGA2), and type IV collagen α3 (COL4A3). Loss of both collagen receptors DDR1 and integrin α2β1 delays maturation of the GBM: due to a porous GBM filtration barrier high molecular weight proteinuria that more than doubles between day 60 and day 100. Thereafter, maturation of the GBM causes proteinuria to drop down to one tenth until day 200. Proteinuria and the porous GBM cause accumulation of glomerular and tubulointerstitial matrix, which both decrease significantly after GBM-maturation until day 250. In parallel, in a disease with impaired GBM-maturation such as Alport syndrome, loss of integrin α2β1 positively delays renal fibrosis: COL4A3^−/−/ITGA2^−/− double knockouts exhibited reduced proteinuria and urea nitrogen compared to COL4A3^−/−/ITGA2^+/− and COL4A3^−/−/ITGA2^+/+ mice. The double knockouts lived 20% longer and showed less glomerular and tubulointerstitial extracellular matrix deposition than the COL4A3^−/− Alport mice with normal integrin α2β1 expression. Electron microscopy illustrated improvements in the glomerular basement membrane structure. MMP2, MMP9, MMP12 and TIMP1 were expressed at significantly higher levels (compared to wild-type mice) in COL4A3^−/−/ITGA2^+/+ Alport mice, but not in COL4A3^+/+/ITGA2^−/− mice. In conclusion, the collagen receptors DDR1 and integrin α2β1 contribute to regulate GBM-maturation and to control matrix accumulation. As demonstrated in the type IV collagen disease Alport syndrome, glomerular cell–matrix interactions via collagen receptors play an important role in the progression of renal fibrosis.     

Transferrin receptor 2 mitigates periodontitis-driven alveolar bone loss

Periodontitis is associated with significant alveolar bone loss. Patients with iron overload suffer more frequently from periodontitis, however, the underlying mechanisms remain largely elusive. Here, we investigated the role of transferrin receptor 2 (Tfr2), one of the main regulators of iron homeostasis, in the pathogenesis of periodontitis and the dental phenotype under basal conditions in mice. As Tfr2 suppresses osteoclastogenesis, we hypothesized that deficiency of Tfr2 may exacerbate periodontitis-induced bone loss. Mice lacking Tfr2 (Tfr2^−/−) and wild-type (Tfr2^+/+) littermates were challenged with experimental periodontitis. Mandibles and maxillae were collected for microcomputed tomography and histology analyses. Osteoclast cultures from Tfr2^+/+ and Tfr2^−/− mice were established and analyzed for differentiation efficiency, by performing messenger RNA expression and protein signaling pathways. After 8 days, Tfr2-deficient mice revealed a more severe course of periodontitis paralleled by higher immune cell infiltration and a higher histological inflammation index than Tfr2^+/+ mice. Moreover, Tfr2-deficient mice lost more alveolar bone compared to Tfr2^+/+ littermates, an effect that was only partially iron-dependent. Histological analysis revealed a higher number of osteoclasts in the alveolar bone of Tfr2-deficient mice. In line, Tfr2-deficient osteoclastic differentiation ex vivo was faster and more efficient as reflected by a higher number of osteoclasts, a higher expression of osteoclast markers, and an increased resorptive activity. Mechanistically, Tfr2-deficient osteoclasts showed a higher p38-MAPK signaling and inhibition of p38-MAPK signaling in Tfr2-deficient cells reverted osteoclast formation to Tfr2^+/+ levels. Taken together, our data indicate that Tfr2 modulates the inflammatory response in periodontitis thereby mitigating effects on alveolar bone loss.     

Browning deficiency and low mobilization of fatty acids in gonadal white adipose tissue leads to decreased cold-tolerance of transglutaminase 2 knock-out mice

During cold-exposure ‘beige’ adipocytes with increased mitochondrial content are activated in white adipose tissue (WAT). These cells, similarly to brown adipose tissue (BAT), dissipate stored chemical energy in the form of heat with the help of uncoupling protein 1 (UCP1). We investigated the effect of tissue transglutaminase (TG2) ablation on the function of ATs in mice. Although TG2^+/+ and TG2^−/− mice had the same amount of WAT and BAT, we found that TG2^+/+ animals could tolerate acute cold exposure for 4 h, whereas TG2^−/− mice only for 3 h. Both TG2^−/− and TG2^+/+ animals used up half of the triacylglycerol content of subcutaneous WAT (SCAT) after 3 h treatment; however, TG2^−/− mice still possessed markedly whiter and higher amount of gonadal WAT (GONAT) as reflected in the larger size of adipocytes and lower free fatty acid levels in serum. Furthermore, lower expression of ‘beige’ marker genes such as UCP1, TBX1 and TNFRFS9 was observed after cold exposure in GONAT of TG2^−/− mice, paralleled with a lower level of UCP1 protein and a decreased mitochondrial content. The detected changes in gene expression of Resistin and Adiponectin did not provoke glucose intolerance in the investigated TG2^−/− mice, and TG2 deletion did not influence adrenaline, noradrenaline, glucagon and insulin production. Our data suggest that TG2 has a tissue-specific role in GONAT function and browning, which becomes apparent under acute cold exposure.     

Role of Pituitary Adenylate-Cyclase Activating Polypeptide and Tac1 gene derived tachykinins in sensory,motor and vascular functions under normal and neuropathic conditions

Pituitary Adenylate-Cyclase Activating Polypeptide (PACAP) and Tac1 gene-encoded tachykinins (substance P: SP, neurokinin A: NKA) are expressed in capsaicin-sensitive nerves, but their role in nociception, inflammation and vasoregulation is unclear. Therefore, we investigated the function of these neuropeptides and the NK₁ tachykinin receptor (from Tacr1 gene) in the partial sciatic nerve ligation-induced traumatic mononeuropathy model using gene deficient (PACAP^−/−, Tac1^−/−, and Tacr1^−/−) mice. Mechanonociceptive threshold of the paw was measured with dynamic plantar aesthesiometry, motor coordination with Rota-Rod and cutaneous microcirculation with laser Doppler imaging. Neurogenic vasodilation was evoked by mustard oil stimulating sensory nerves. In wildtype mice 30–40% mechanical hyperalgesia developed one week after nerve ligation, which was not altered in Tac1^−/− and Tacr1^−/− mice, but was absent in PACAP^−/− animals. Motor coordination of the PACAP^−/− and Tac1^−/− groups was significantly worse both before and after nerve ligation compared to their wildtypes, but it did not change in Tacr1^−/− mice. Basal postoperative microcirculation on the plantar skin of PACAP^−/− mice did not differ from the wildtypes, but was significantly lower in Tac1^−/− and Tacr1^−/− ones. In contrast, mustard oil-induced neurogenic vasodilation was significantly smaller in PACAP^−/− mice, but not in Tacr1^−/− and Tac1^−/− animals. Both PACAP and SP/NKA, but not NK₁ receptors participate in normal motor coordination. Tachykinins maintain basal cutaneous microcirculation. PACAP is a crucial mediator of neuropathic mechanical hyperalgesia and neurogenic vasodilation. Therefore identifying its target and developing selective, potent antagonists, might open promising new perspectives for the treatment of neuropathic pain and vascular complications.     

Ocular prostaglandin production and morphology in mice lacking a single isoform of cyclooxygenase

Prostaglandins have many important roles in ocular physiology and are used clinically for the treatment of glaucoma. The aim of this study was to analyse the contribution of each cyclooxygenase isoform to ocular prostaglandin production using isoform-specific knockout mice. Ex vivo PGE₂, 6-keto-PGF_1α, and TXB₂ production was measured from whole eyes, corneal tissue, uveoscleral tissue, lens, retina and optic nerve using enzyme-linked immunosorbant assays. Ocular immunohistochemical and histological analysis was also conducted for each genotype. Levels of each of the prostaglandins measured were significantly decreased in the corneal tissue, uveoscleral tissue, lens, retina and optic nerve of COX-1^−/− mice in comparison with wild-type mice. In contrast, COX-2^−/− mice had similar levels of ocular prostaglandin production to wild-type mice. These results suggest that COX-1 is the principal isoform responsible for prostaglandin production in the mouse eye. The absence of COX-1 or COX-2 did not appear to effect ocular development in these mice.     

Remarkable induction of UV-signature mutations at the 3′-cytosine of dipyrimidine sites except at 5′-TCG-3′ in the UVB-exposed skin epidermis of xeroderma pigmentosum variant model mice

The human POLH gene is responsible for the variant form of xeroderma pigmentosum (XP-V), a genetic disease highly susceptible to cancer on sun-exposed skin areas, and encodes DNA polymerase η (polη), which is specialized for translesion DNA synthesis (TLS) of UV-induced DNA photolesions. We constructed polη-deficient mice transgenic with lacZ mutational reporter genes to study the effect of Polh null mutation (Polh^−/−) on mutagenesis in the skin after UVB irradiation. UVB induced lacZ mutations with remarkably higher frequency in the Polh^−/− epidermis and dermis than in the wild-type (Polh^+/+) and heterozygote. DNA sequences of a hundred lacZ mutants isolated from the epidermis of four UVB-exposed Polh^−/− mice were determined and compared with mutant sequences from irradiated Polh^+/+ mice. The spectra of the mutations in the two genotypes were both highly UV-specific and dominated by C → T transitions at dipyrimidines, namely UV-signature mutations. However, sequence preferences of the occurrence of UV-signature mutations were quite different between the two genotypes: the mutations occurred at a higher frequency preferentially at the 5′-TCG-3′ sequence context than at the other dipyrimidine contexts in the Polh^+/+ epidermis, whereas the mutations were induced remarkably and exclusively at the 3′-cytosine of almost all dipyrimidine contexts with no preference for 5′-TCG-3′ in the Polh^−/− epidermis. In addition, in Polh^−/− mice, a small but remarkable fraction of G → T transversions was also observed exclusively at the 3′-cytosine of dipyrimidine sites, strongly suggesting that these transversions resulted not from oxidative damage but from UV photolesions. These results would reflect the characteristics of the error-prone TLS functioning in the bypass of UV photolesions in the absence of polη, which would be mediated by mechanisms based on the two-step model of TLS. On the other hand, the deamination model would explain well the mutation spectrum in the Polh^+/+ genotype.     

Cathelicidin represents a new target for manipulation of skin inflammation in Netherton syndrome

Netherton syndrome (NS) is a severe ichthyosis caused by inactivating mutations in the SPINK5 gene encoding the serine protease inhibitor LEKTI. Spink5^−/− mice recapitulate NS and die perinatally from extensive dehydration as a result of a severe defect of the epidermal barrier. We showed that deletion of Klk5 in Spink5^−/− rescues neonatal lethality (Furio et al., 2015). However, Spink5^−/−Klk5^−/− mice developed skin shedding and inflammation during the first week from birth and the majority (70%) succumbed on P7. The remaining mice lived short (i.e. mean survival was 5 months) indicating alternative inflammatory pathways. Since cathelicidin is increased in Spink5^−/− epidermis, we investigated whether it could be implicated in NS pathology. Ablation of Camp in Spink5^−/− suppressed epidermal inflammation and restored abnormal epidermal differentiation, nevertheless, it failed to inhibit overdesquamation and Spink5^−/−Camp^−/− succumbed perinatally due to skin barrier defect, similarly to Spink5^−/−. Joint invalidation of Klk5 and Camp significantly extended survival of Spink5^−/−Klk5^−/−Camp^−/− mice. We provide evidence that cathelicidin is implicated in NS-associated skin inflammation in vivo. Therefore, marketed products that are known to reduce cathelicidin expression could be repurposed for the management of NS.     

Loss of Nrf2 in bone marrow-derived macrophages impairs antigen-driven CD8+ T cell function by limiting GSH and Cys availability

NF-E2-related factor 2 (Nrf2), known to protect against reactive oxygen species, has recently been reported to resolve acute inflammatory responses in activated macrophages. Consequently, disruption of Nrf2 promotes a proinflammatory macrophage phenotype. In the current study, we addressed the impact of this macrophage phenotype on CD8⁺ T cell activation by using an antigen-driven coculture model consisting of Nrf2^−/− and Nrf2^+/+ bone marrow-derived macrophages (BMDMΦ) and transgenic OT-1 CD8⁺ T cells. OT-1 CD8⁺ T cells encode a T cell receptor that specifically recognizes MHC class I-presented ovalbumin OVA(257–264) peptide, thereby causing a downstream T cell activation. Interestingly, coculture of OVA(257–264)-pulsed Nrf2^−/− BMDMΦ with transgenic OT-1 CD8⁺ T cells attenuated CD8⁺ T cell activation, proliferation, and cytotoxic function. Since the provision of low-molecular-weight thiols such as glutathione (GSH) or cysteine (Cys) by macrophages limits antigen-driven CD8⁺ T cell activation, we quantified the amounts of intracellular and extracellular GSH and Cys in both cocultures. Indeed, GSH levels were strongly decreased in Nrf2^−/− cocultures compared to wild-type counterparts. Supplementation of thiols in Nrf2^−/− cocultures via addition of glutathione ester, N-acetylcysteine, β-mercaptoethanol, or cysteine itself restored T cell proliferation as well as cytotoxicity by increasing intracellular GSH. Mechanistically, we identified two potential Nrf2-regulated genes involved in thiol synthesis in BMDMΦ: the cystine transporter subunit xCT and the modulatory subunit of the GSH-synthesizing enzyme γ-GCS (GCLM). Pharmacological inhibition of γ-GCS-dependent GSH synthesis as well as knockdown of the cystine antiporter xCT in Nrf2^+/+ BMDMΦ mimicked the effect of Nrf2^−/− BMDMΦ on CD8⁺ T cell function. Our findings demonstrate that reduced levels of GCLM as well as xCT in Nrf2^−/− BMDMΦ limit GSH availability, thereby inhibiting antigen-induced CD8⁺ T cell function.     

Eicosanoid pathway on host resistance and inflammation during Mycobacterium tuberculosis infection is comprised by LTB4 reduction but not PGE2 increment

The functions of eicosanoids, a family of potent biologically active lipid mediators, are not restricted to inflammatory responses and they also act as mediators of the pathogenesis process. However, the role of eicosanoids in tuberculosis remains controversial. To investigate the specific role of LTB₄ in Mycobacterium tuberculosis (Mtb) infection, we used 5-lipoxygenase-deficient (5-LO^−/−) mice and WT (sv129) mice inoculated intranasally with LTB₄ (encapsulated in PLGA microspheres). We showed that deficiency of the 5-LO pathway was related to resistance to Mtb infection. LTB₄ inoculation increased susceptibility to Mtb in 5-LO^−/− mice but not in WT mice, resulting in worsening of lung inflammation and tissue damage. In infected WT mice, most supplementary LTB₄ was metabolized to the inactive form 12-oxo-LTB₄ in the lung. A high amount of PGE₂ was detected during Mtb infection, and pharmacological inhibition of COX-2 induced a significant reduction of bacterial load and an improved innate immune response in the lungs, independently of baseline LTB₄ levels. COX-2 inhibition with celecoxib significantly reduced PGE₂ levels, enhanced IFN-γ production and NO release, and increased macrophage phagocytosis of Mtb. The results suggest that a balance between PGE₂/LTB₄ is essential in the pathogenesis process of tuberculosis to prevent severe inflammation. Moreover, optimal levels of PGE₂ are required to induce an effective innate response in the early phase of Mtb infection. Thus, pharmacological modulation of eicosanoid production may provide an important host-directed therapy in tuberculosis.     

The circadian gene Cryptochrome 2 influences stress-induced brain activity and depressive-like behavior in mice

Cryptochrome 2 (Cry2) is a core clock gene important for circadian regulation. It has also been associated with anxiety and depressive-like behaviors in mice, but the previous findings have been conflicting in terms of the direction of the effect. To begin to elucidate the molecular mechanisms of this association, we carried out behavioral testing, PET imaging, and gene expression analysis of Cry2^−/− and Cry2^+/+ mice. Compared to Cry2^+/+ mice, we found that Cry2^−/− mice spent less time immobile in the forced swim test, suggesting reduced despair-like behavior. Moreover, Cry2^−/− mice had lower saccharin preference, indicative of increased anhedonia. In contrast, we observed no group differences in anxiety-like behavior. The behavioral changes were accompanied by lower metabolic activity of the ventro-medial hypothalamus, suprachiasmatic nuclei, ventral tegmental area, anterior and medial striatum, substantia nigra, and habenula after cold stress as measured by PET imaging with a glucose analog. Although the expression of many depression-associated and metabolic genes was upregulated or downregulated by cold stress, we observed no differences between Cry2^−/− and Cry2^+/+ mice. These findings are consistent with other studies showing that Cry2 is required for normal emotional behavior. Our findings confirm previous roles of Cry2 in behavior and extend them by showing that the effects on behavior may be mediated by changes in brain metabolism.     

Cyclooxygenase-2 in newborn hyperoxic lung injury

Supraphysiological O₂ concentrations, mechanical ventilation, and inflammation significantly contribute to the development of bronchopulmonary dysplasia (BPD).Exposure of newborn mice to hyperoxia causes inflammation and impaired alveolarization similar to that seen in infants with BPD.Previously, we demonstrated that pulmonary cyclooxygenase-2 (COX-2) protein expression is increased in hyperoxia-exposed newborn mice.The present studies were designed to define the role of COX-2 in newborn hyperoxic lung injury.We tested the hypothesis that attenuation of COX-2 activity would reduce hyperoxia-induced inflammation and improve alveolarization.Newborn C3H/HeN micewere injected daily with vehicle, aspirin (nonselective COX-2 inhibitor), or celecoxib (selective COX-2 inhibitor) for the first 7 days of life.Additional studies utilized wild-type (C57Bl/6, COX-2^+/+), heterozygous (COX-2^+/-), and homozygous (COX-2^-/-) transgenic mice.Micewere exposed to room air (21% O₂) or hyperoxia (85% O₂) for 14 days.Aspirin-injected and COX-2^-/- pups had reduced levels of monocyte chemoattractant protein (MCP-1) in bronchoalveolar lavage fluid (BAL).Both aspirin and celecoxib treatment reduced macrophage numbers in the alveolar walls and air spaces.Aspirin and celecoxib treatment attenuated hyperoxia-induced COX activity, including altered levels of prostaglandin (PG)D₂ metabolites.Decreased COX activity, however, did not prevent hyperoxia-induced lung developmental deficits.Our data suggest thatincreased COX-2 activity may contribute to proinflammatory responses, including macrophage chemotaxis, during exposure to hyperoxia.Modulation of COX-2 activity may be a useful therapeutic target to limit hyperoxia-induced inflammation in preterm infants at risk of developing BPD.