Search

Multicellular aggregates have been widely utilized for regenerative medicine; however, the heterogeneous structure and undesired bioactivity of cell-only aggregates hinder their clinical translation. In this study, we fabricated an innovative kind of microparticle-integrated cellular aggregate with multifunctional activities in angiogenesis and osteogenesis, by combining stem cells from human exfoliated deciduous teeth (SHEDs) and bioactive composite microparticles. The poly(lactide

A public health crisis in the form of a significant incidence of fatal pulmonary disease caused by repeated use of humidifier disinfectants containing polyhexamethylene guanidine phosphate (PHMG) recently arose in Korea. Although the mechanisms of pulmonary fibrosis following respiratory exposure to PHMG are well described, distant-organ effect has not been reported. In this study, we investigated whether intratracheal administration of PHMG affects liver pathophysiology and metabolism. Our PHMG mouse model showed a significant decrease in liver cholesterol level. An mRNA-seq analysis of liver samples revealed an alteration in the gene expression associated with cholesterol biosynthesis and metabolism to bile acids. The expression of genes involved in cholesterol synthesis was decreased in a real-time PCR analysis. To our surprise, we found that the coordinate regulation of cholesterol and bile acid homeostasis was completely disrupted. Despite the decreased cholesterol synthesis and low bile acid levels, the farnesoid X receptor/small heterodimer partner pathway, which controls negative feedback of bile acid synthesis, was activated in PHMG mice. As a consequence, gene expression of Cyp7a1 and Cyp7b1, the rate-limiting enzymes of the classical and alternative pathways of bile acid synthesis, was significantly downregulated. Notably, the changes in gene expression were corroborated by the hepatic concentrations of the bile acids. These results suggest that respiratory exposure to PHMG could cause cholestatic liver injury by disrupting the physiological regulation of hepatic cholesterol and bile acid homeostasis.

Alphaherpesviruses are large dsDNA viruses with an ability to establish persistent infection in hosts, which rely partly on their ability to evade host innate immune responses, notably the type I IFN response. However, the relevant molecular mechanisms are not well understood. In this study, we report the UL42 proteins of alphaherpesvirus pseudorabies virus (PRV) and HSV type 1 (HSV1) as a potent antagonist of the IFN-I–induced JAK-STAT signaling pathway. We found that ectopic expression of UL42 in porcine macrophage CRL and human HeLa cells significantly suppresses IFN-α–mediated activation of the IFN-stimulated response element (ISRE), leading to a decreased transcription and expression of IFN-stimulated genes (ISGs). Mechanistically, UL42 directly interacts with ISRE and interferes with ISG factor 3 (ISGF3) from binding to ISRE for efficient gene transcription, and four conserved DNA-binding sites of UL42 are required for this interaction. The substitution of these DNA-binding sites with alanines results in reduced ISRE-binding ability of UL42 and impairs for PRV to evade the IFN response. Knockdown of UL42 in PRV remarkably attenuates the antagonism of virus to IFN in porcine kidney PK15 cells. Our results indicate that the UL42 protein of alphaherpesviruses possesses the ability to suppress IFN-I signaling by preventing the association of ISGF3 and ISRE, thereby contributing to immune evasion. This finding reveals UL42 as a potential antiviral target.

The c-MYB transcription factor is a key regulator of hematopoietic cell proliferation and differentiation, and dysregulation of c-MYB activity often associates with various hematological disorders. Yet, its pathogenic role remains largely unknown due to lack of suitable animal models. Here, we report a detail characterization of a c-myb-gfp transgenic zebrafish harboring c-Myb hyperactivity (named c-mybhyper). This line exhibits abnormal granulocyte expansion that resembles human myelodysplastic syndrome (MDS) from embryonic stage to adulthood. Strikingly, a small portion of c-mybhyper adult fish develops acute myeloid leukemia-like or acute lymphoid leukemia-like disorders with age. The myeloid and lymphoid malignancies in c-mybhyper adult fish are likely caused by the hyperactivity of c-myb, resulting in the dysregulation of a number of cell-cycle-related genes and hyperproliferation of hematopoietic precursor cells. Finally, treatment with c-myb target drug flavopiridol can relieve the MDS-like symptoms in both c-mybhyper embryos and adult fish. Our study establishes a zebrafish model for studying the cellular and molecular mechanisms underlying c-Myb-associated leukemogenesis as well as for anti-leukemic drug screening.

Oral Diseases (2010) 16, 351–359 Objectives: To investigate the changes in the muscarinic receptor signaling pathway with submandibular gland (SMG) transplantation and whether carbachol improves secretion in transplanted SMGs. Materials and methods: SMG autotransplantation was performed in a rabbit model. Carbachol (1 μM) was infused into the transplanted glands from postoperative day 1–7. The expression of the M1 and M3 muscarinic receptors, aquaporin-5 (AQP5), and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was measured by RT-PCR, immunoblotting or immunofluorescence. The content of inositol 1, 4, 5-trisphosphate (IP3) was measured by radioimmunoassay. Results: Salivary flow of the transplanted SMGs was decreased after transplantation. As well, the expressions of M1 and M3 receptors and their downstream signaling molecules, IP3, p-ERK1/2 and AQP5, were all reduced. Atrophy of acinar cells was shown in transplanted glands. However, all these alterations were reversed after carbachol treatment for 7 days. Furthermore, carbachol directly increased the mRNA expression of AQP5 and phosphorylation of ERK1/2 in cultured neonatal rabbit SMG cells. Conclusion: A lack of acetylcholine and downregulation of the muscarinic receptor signaling pathway is involved in the early hypofunction of transplanted SMGs. Carbachol treatment could be a new therapeutic strategy to improve secretion and prevent the obstruction of Wharton’s duct in the early phase after SMG transplantation.

Defensins play an important role in both innate and adaptive immunity due to their antimicrobial, regulatory, and chemotactic effects. Nonetheless, the role of murine β-defensins (mBD) 3 and 4, the murine homologs of human β-defensins (hBD) 2 and 3, remains unknown in Pseudomonas aeruginosa keratitis. This study explored their role in corneal infection and potential synergy with mBD2, a defensin associated with better outcome in this disease. Immunostaining and real-time RT-PCR data demonstrated that mBD3 and mBD4 expression was inducible and differentially regulated in the infected cornea of resistant BALB/c vs susceptible C57BL/6 (B6) mice. Knockdown studies using small interfering RNA treatment indicated that mBD3, but not mBD4, is required in ocular defense. Moreover, in vivo studies demonstrated individual and combined effects of mBD2 and mBD3 that modulate bacterial load, polymorphonuclear neutrophil (PMN) infiltration, and production of IFN-γ, MIP-2, IL-1β, TNF-α, inducible NO synthase (iNOS), TLR2, TLR4, MyD88, and NF-κB. Most notably, bacterial load was increased at 5 days postinfection by silencing either mBD2 or mBD3, but it was elevated at both 1 and 5 days postinfection when silencing both defensins. PMN infiltration was increased at 1 day postinfection by silencing both defensins or mBD3, but not mBD2 alone. iNOS expression was elevated by silencing mBD2, but it was reduced after silencing mBD3 or both defensins. Additionally, cell sources of mBD2 (macrophages, PMN and fibroblasts) and mBD3 (PMN) in corneal stroma were identified by dual label immunostaining after infection. Collectively, the data provide evidence that mBD2 and mBD3 together promote resistance against corneal infection.

Objective To investigate a possible role of adiponectin in the pathogenesis of autoimmune sialoadenitis in non-obese diabetic (NOD) mouse model of Sjogren's syndrome. Materials and Methods Expression of adiponectin and its receptors (AdipoR1/2) was detected by PCR, immunoblotting, or immunofluorescence. The level of adiponectin was quantified by ELISA. Adiponectin-related signaling molecules and pro-inflammatory cytokines were examined by PCR or immunoblotting. Apoptosis was evaluated by TUNEL staining, flow cytometry, and caspase 3 activation. Results Adiponectin and AdipoR1/2 mRNA and protein were expressed in submandibular glands. Adiponectin immunostaining was widely diffused in the cytoplasm of acinar and ductal cells. AdipoR1 was mainly distributed in acinar cytoplasm, while AdipoR2 was predominantly located at acinar cell membrane. Submandibular adiponectin levels were reduced during the progression of autoimmune sialoadenitis in 7-, 14-, and 21-week-old NOD mice, while AdipoR1/2 levels were unchanged. The levels of phosphorylated adenosine monophosphate-activated protein kinase, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase were decreased, while interferon (IFN)-γ and glandular apoptosis were temporally increased at all time points. Moreover, exogenous adiponectin supplement inhibited, whereas neutralizing endogenous adiponectin by its antibody promoted IFN-γ-induced apoptosis and caspase 3 activation in cultured submandibular acinar cells. Conclusions Adiponectin plays a protective role on submandibular cells. Decreased adiponectin might promote glandular destruction in autoimmune sialoadenitis.

This study analyzed the influence of substance P (SP) on growth factors related to wound healing in mice in the presence of infectious keratitis.Naturally resistant mice were injected intraperitoneally with SP or phosphate-buffered saline and infected with Pseudomonas aeruginosa, and corneal messenger RNA (mRNA) levels of growth factors and apoptosis genes were tested. Enzyme-linked immunosorbent assay determined the protein levels, whereas immunohistochemistry tested the distribution, macrophage phenotype, and cell quantitation. In vitro, macrophages were stimulated with lipopolysaccharide (LPS; with or without SP) and mRNA levels of proinflammatory and antiinflammatory cytokines and apoptosis genes were tested.After SP, epidermal growth factor mRNA and protein levels were disparately regulated early, with no differences later in the disease. Hepatocyte growth factor and fibroblast growth factor-7 mRNA and protein levels were increased after SP treatment. Enumerating dual-labeled stromal cells revealed no difference between SP-treated versus phosphate-buffered saline-treated groups in the percentage of epidermal growth factor-labeled fibroblasts or macrophages, but there were significant increases in both hepatocyte growth factor- and fibroblast growth factor-7-labeled cells. Type 2 (M2) macrophages and caspase-3 mRNA levels were decreased, whereas B-cell lymphoma-2 mRNA expression was increased after SP treatment. In vitro, mRNA levels of several proinflammatory cytokines and B-cell lymphoma-2 were elevated, whereas transforming growth factor β was decreased after macrophage stimulation with SP (with LPS) over LPS alone. (Mice: n = 105 control; 105 experimental.)These data show that treatment with SP in infectious keratitis elevates growth factors but also adversely affects the disease by enhancing the inflammatory response and its sequelae.

TLRs recognize microbial pathogens and trigger an immune response, but their regulation by neuropeptides, such as vasoactive intestinal peptide (VIP), during Pseudomonas aeruginosa corneal infection remains unexplored. Therefore, C57BL/6 (B6) mice were injected i.p. with VIP, and mRNA, protein, and immunostaining assays were performed. After VIP treatment, PCR array and real-time RT-PCR demonstrated that proinflammatory TLRs (conserved helix-loop-helix ubiquitous kinase, IRAK1, TLR1, TLR4, TLR6, TLR8, TLR9, and TNFR-associated factor 6) were downregulated, whereas anti-inflammatory TLRs (single Ig IL-1–related receptor [SIGIRR] and ST2) were upregulated. ELISA showed that VIP modestly downregulated phosphorylated inhibitor of NF-κB kinase subunit α but upregulated ST2 ~2-fold. SIGIRR was also upregulated, whereas TLR4 immunostaining was reduced in cornea; all confirmed the mRNA data. To determine whether VIP effects were cAMP dependent, mice were injected with small interfering RNA for type 7 adenylate cyclase (AC7), with or without VIP treatment. After silencing AC7, changes in mRNA levels of TLR1, TNFR-associated factor 6, and ST2 were seen and unchanged with addition of VIP, indicating that their regulation was cAMP dependent. In contrast, changes were seen in mRNA levels of conserved helix-loop-helix ubiquitous kinase, IRAK1, 2, TLR4, 9 and SIGIRR following AC7 silencing alone; these were modified by VIP addition, indicating their cAMP independence. In vitro studies assessed the effects of VIP on TLR regulation in macrophages and Langerhans cells. VIP downregulated mRNA expression of proinflammatory TLRs while upregulating anti-inflammatory TLRs in both cell types. Collectively, the data provide evidence that VIP downregulates proinflammatory TLRs and upregulates anti-inflammatory TLRs and that this regulation is both cAMP dependent and independent and involves immune cell types found in the infected cornea.

Keratitis caused by Pseudomonas aeruginosa is characterized by epithelial edema, stromal infiltrate, and corneal ulceration and can lead to vision loss.1 This sight-threatening infectious disease is, in large part, a consequence of the host inflammatory response,2 with previous studies showing that both innate and adaptive host immune responses are critical in its development.3,4 The innate host response to bacterial infection is primarily mediated by polymorphonuclear neutrophils (PMN) and macrophages (Mφ).5 The initial phase of host defense against many invading microbes such as P. aeruginosa also involves a family of proteins called Toll-like receptors (TLRs), which recognize microbial products and trigger an innate immune response,6,7 leading to the expression of various pro-inflammatory and anti-inflammatory cytokines/chemokines,8 including tumor necrosis factor alpha (TNF-α), interferon-gamma (IFN-γ), and macrophage inflammatory protein (MIP)-2, interleukin (IL)-1β, -4, -5, -6, -10, and -12. Inflammatory mediators may promote the elimination of bacteria, but, if they are unbalanced or uncontrolled, they may augment the inflammatory response, leading to tissue damage and corneal perforation. For example, gene expression profiling of Mφ has shown that Gram-negative bacteria induce transcriptional activation of a common host response that induces genes in Mφ, expressing an M1 program.9 M1 polarized cells, prototypical in Th1 responder strains of mice, such as B6,10 are characterized by the production of IL-12, TNF-α, MIP-2, and high levels of nitric oxide synthase 2 (NOS2).11,12 Excessive or prolonged M1 polarization can lead to tissue injury and contribute to pathogenesis. In contrast, Th2 responder mice (BALB/c) have a higher population of alternatively activated Mφ, designated M2 cells, that produce anti-inflammatory mediators such as IL-10, IL-1ra, and type II IL-1 decoy receptor,12 upregulate the production of arginase 1 (Arg1),10 and are critical to disease resolution. A subset of the latter cells can be induced by agonists of TLRs, including LPS.9 Thus, negative regulation of TLR signaling may be critical to avoid a detrimental and inappropriate inflammatory response.13 In this regard, the soluble TLRs (sTLR2 and sTLR4) act as decoy receptors by binding to their ligands and competitively blocking TLR2 and TLR4 signaling,14 whereas the IL-1 receptor–related protein ST2 negatively regulates TLR signaling by sequestering the recruitment of adaptor molecules such as MyD88 and TIRAP.15 ST2 is a novel member of the TLR superfamily with unique anti-inflammatory properties. ST2 mRNAs were expressed in all human tissues examined, induced by cytokines and phorbol esters. Three species of mRNAs were observed in different human cells and tissues. In contrast, only two species of ST2 mRNAs were observed in BALB/c-3T3 cells, and ST2 mRNA was absent in most tissues of normal mice.16 Higher expression levels on the surfaces of fibroblasts,17 mast cells,18 and Th2 cells19,20 have been reported. IL-33 is a recently identified member of the IL-1 family that signals through ST2. The interaction between IL-33 and ST2 mediate its biological effects by recruiting the adaptor molecules MyD88, IRAK1, IRAK4, and TRAF6, activating mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB, leading to the production of Th2-associated cytokines such as IL-4, -5, and -13. IL-33 is most closely related structurally to the IL-1 family, including IL-1β and IL-18, and has important functions in host defense, immune regulation, and inflammation.21 However, unlike IL-1β and IL-18, which promote pro-inflammatory and Th1-associated responses, IL-33 predominantly induces the production of Th2 cytokines and increases levels of serum immunoglobulin.21 In vitro, IL-33 enhanced IL-5 and IL-13 production by polarized Th2, but not Th1, cell lines.22 In addition, the in vivo administration of exogenous IL-33 into naive mice provoked type 2 responses, production of Th2 cytokines and IgE, eosinophilia, and some pathologic changes in mucosal tissues.21 Although IL-33 was detected in epithelial cells from the bronchus and small airways, fibroblasts, and smooth muscle cells, which suggested a possible role in the regulation of mucosal inflammation,23,24 the expression and functional role of IL-33 (as a specific ligand of ST2) in bacterial keratitis remains unknown. In the present study, we tested the expression of IL-33 in the cornea of B6 and BALB/c mice before and after P. aeruginosa infection. Our data provide direct evidence that IL-33 is constitutively expressed in the cornea (epithelium) and is significantly elevated in BALB/c over B6 mice after infection. Furthermore, B6 mice treated with rmIL-33 protein had less severe corneal disease after P. aeruginosa infection. Mechanistically, our data support the tenets that treatment reduced corneal infection and inflammation by negatively regulating pro-inflammatory cytokines, polarizing corneal Mφ to produce anti-inflammatory mediators such as Arg1, and upregulating type 2 and anti-inflammatory cytokine production.

OBJECTIVES This study used a rat contact lens (CL) model to test if high- versus low-Dk lens wear caused changes in (1) conjunctival Langerhans cell (LC) number or location; (2) Bcl-2 expression; and (3) infection risk. METHODS Female, Lewis rats wore a high- or low-Dk CL continuously for 2 weeks. Afterward, corneas were harvested and processed for ADPase activity to identify LCs, for immunostaining and for real time-polymerase chain reaction. Contact lens-wearing rats also were challenged with Pseudomonas aeruginosa by placing a bacterial-soaked CL on the eye followed by topical delivery of bacteria. After 48 hrs, slit lamp examination and real time-polymerase chain reaction were used to evaluate the corneal response. RESULTS Conjunctival LC were significantly increased after low- versus high-Dk CL wear (P

PURPOSE: Apoptosis was examined after Pseudomonas aeruginosa corneal infection in C57BL/6 (B6, susceptible) and BALB/c (resistant) mice. METHODS: TUNEL staining, real-time RT-PCR, polymorphonuclear neutrophils (PMNs) and macrophage (Mphi) depletion, and immunostaining were used. RESULTS: Intense TUNEL staining was seen in BALB/c versus B6 cornea at 1 versus 3 days after infection (PI) and correlated with mRNA levels for caspase-3. TUNEL staining (with or without PMN depletion) and PMN immunostaining revealed the PMN as the major apoptotic cell for both groups. Next, B6 mice with high corneal levels of the antiapoptosis neuropeptide, substance P (SP), were treated with the SP antagonist, Spantide I (with/without Mphi depletion), resulting in earlier apoptosis and diminished disease only when M(phi)s were present. SP interactions with M(phi)s were explored further by eliciting cells from both groups and stimulating them with lipopolysaccharide (LPS), with or without SP. LPS with SP treatment decreased the number of apoptotic M(phi)s in B6 but not BALB/c mice and correlated with reduced mRNA expression of NK-1R (major SP receptor) on BALB/c cells. In addition, mRNA expression for IL-12 was upregulated in LPS-stimulated B6 M(phi)s, although cells from BALB/c mice expressed more IL-10. CONCLUSIONS: These studies provide evidence that PMN apoptosis is delayed in the cornea of B6 versus BALB/c mice after bacterial infection; that in B6 mice, blocking SP interaction with the NK-1R promotes earlier apoptosis and improves disease outcome; that M(phi)s regulate PMN apoptosis; and that M(phi)s from B6 versus BALB/c mice differ in expression of the NK-1R and cytokines produced after LPS challenge.

Purpose To determine the effects of blocking substance P (SP) interactions with its major receptor (NK1-R) using the antagonist spantide I in susceptible mice infected with Pseudomonas aeruginosa. Methods Immunohistochemistry and enzyme immunosorbent assay (EIA) tested levels of SP in the cornea of B6 and BALB/c mice. B6 mice were treated with spantide, and after infection, slit lamp examination; clinical score; bacterial counts; and myeloperoxidase (MPO), RT-PCR, ELISA, and polymorphonuclear (PMN) cell chemotaxis assays were performed. Results SP corneal levels were significantly elevated constitutively and after infection in the B6 more than in BALB/c mice. Spantide treatment of B6 mice significantly decreased the number of perforated corneas, bacterial counts, and PMNs. mRNA levels for type I cytokines (e.g., IFN-gamma) as well as MIP-2, IL-6, TNF-alpha, and IL-1beta (mRNA and protein) also were significantly reduced after spantide treatment. The type II cytokine IL-10 (mRNA and protein) was elevated, whereas TGF-beta mRNA levels were unchanged after spantide treatment. PMN chemotaxis was induced by SP and other neuropeptides in vitro, but was not affected by spantide I. mRNA for neurokinin-1-receptor-1 (NK-1R) was detected in the normal and infected corneas and on macrophages (Mphis), but not on PMNs (unstimulated or stimulated with endotoxin [LPS]). Spantide treatment of Mphis reduced IL-1beta after LPS+SP treatment but not after either alone. Conclusions The SP antagonist Spantide provides a novel approach to reduce type 1 and enhance the type 2 cytokine IL-10 in the infected cornea of B6 mice, leading to a significant reduction in corneal perforation and improved disease outcome.

Purpose. To examine the early host response to Pseudomonas aeruginosa challenge in the extended contact lens–wearing rat model. Methods. Lewis rats were fitted with extended-wear lotrafilcon A hydrogel lenses in the left eye, and the right eye served as the control. Bacterial challenge was initiated in the experimental eye by fitting a bacteria-soaked contact lens and by topical delivery of the bacteria. On first detection of corneal opacity, slitlamp examination, histopathologic examination, viable bacteria counts, enzyme-linked immunosorbent assay, myeloperoxidase, Langerhans cell detection, and multiprobe ribonuclease protection assays were used to evaluate the early corneal response. Results. Analysis of bacterially challenged contact lens–wearing versus control rats showed Langerhans cells and polymorphonuclear neutrophils only in the experimentally challenged cornea. In addition, in the experimentally challenged cornea, ribonuclease protection and enzyme-linked immunosorbent analyses showed an upregulation of proinflammatory cytokines, including interleukins 1β and 6, suggesting that with contact lens wear, these cytokines contribute to the early corneal response and, potentially, disease. Conclusions. The contact lens–wearing rat model allows a unique analysis of the early effects of bacterial challenge in extended-wear contact lenses in the absence of corneal scarring, used in most rodent models. The rat model should be valuable to delineate further the effects of contact lens wear, including the testing of additional contact lens–related complications.

Exogenous vasoactive intestinal peptide (VIP) down-regulates pro-inflammatory but up-regulates anti-inflammatory cytokines, growth factors (GFs) and Toll-like receptors promoting healing in experimental Pseudomonas aeruginosa (P. aeruginosa) keratitis. Whether VIP is required for GF or GF receptor (R) expression in normal and infected corneas is unknown and is the purpose of this study.VIP knockout ((-/-)) and wild-type (WT) C57BL/6 (B6) mice were infected and tested using PCR array, real-time RT-PCR, ELISA, and immunostaining. VIP antagonist treatment studies also were done using B6 and BALB/c mice.Infected corneas of VIP(-/-) versus WT B6 mice perforated earlier (2 vs. 5 days postinfection [p.i.]), and array data showed that GFs were differentially changed between groups. RT-PCR revealed that the infected cornea of VIP(-/-) versus WT mice expressed higher mRNA levels of epidermal growth factor (EGF) and hepatocyte growth factor (HGF), reduced FGF, EGFR, and HGFR, with no difference in FGFR; differences between groups were not seen in normal cornea. Immunostaining for GF and GFR in the normal cornea of VIP(-/-) versus WT mice was similar. However, at 1 day p.i., VIP(-/-) versus WT mice had more intense EGF and HGF, similar FGFR, and reduced FGF, EGFR, and HGFR staining. VIP antagonist treatment decreased protein levels for GFR at 5 days p.i. in both B6 and BALB/c mice, with no significant changes in normal cornea.The data showed that endogenous VIP is not requisite for GF or GFR expression in the normal cornea but, after infection, its absence or reduction is critical for their regulation.

An opportunistic, gram-negative pathogen, Pseudomonas aeruginosa is one of the most virulent organisms associated with microbial keratitis and is often associated with contact lens usage.1 Both bacterial (e.g., lipopolysaccharide) and host factors released from infiltrating cells during infection are thought to contribute to a rapidly progressing liquefactive stromal necrosis.2–5 Experimental murine models of the disease have been established: Th1 responder mouse strains (e.g., C57BL/6, B6) are susceptible (cornea perforates), whereas Th2 responder strains (e.g., BALB/c) are resistant (cornea heals).6 In previous studies using these murine models, we provided evidence that an anti-inflammatory neuropeptide, vasoactive intestinal peptide (VIP) given exogenously, promotes resistance against P. aeruginosa corneal infection by regulation of cytokine production and subsequent alteration of the host inflammatory cell response.7 Neuropeptides, such as VIP are small protein-like molecules used by neurons to bidirectionally communicate with the cells of the immune system.8 In the eye, VIP can be detected in corneal nerves and the aqueous humor in both mice and humans.9 Others also have shown previously that VIP reduces pro-inflammatory cytokine production, limits cell-mediated immunity, and inhibits macrophage and T cell proliferation9; and by downregulation of IL-6 and TNF-α, the neuropeptide showed a protective effect in models of endotoxemia.10 Despite these anti-inflammatory effects which have been reported in diverse systems and which clearly modulate disease outcome, the role of VIP in regulation of corneal healing, specifically, control of growth factor production, has not been examined. Classically, growth factors, which also may be considered as cytokines, bind to their receptors and regulate cell growth, proliferation, migration, and differentiation.11 Thus, the current studies investigated the potential regulation by VIP of growth factors in P. aeruginosa-infected corneas in B6 (susceptible) mice that exhibit lower levels of VIP when compared with resistant (BALB/c) mice.7 Exogenous VIP treatment was given to B6 mice to determine whether VIP modulates growth factor production and thereby, favors resolution of disease. Data from these studies provide evidence that VIP treatment leads to upregulation of growth factor production in the cornea after P. aeruginosa infection, and that both epithelial and stromal cells are participatory. Furthermore, evidence is provided to show that treatment with a mixture of growth factors, given topically after infection, prevents perforation of the cornea. Mechanistically this is achieved by downregulation of proinflammatory cytokines, upregulation of anti-inflammatory cytokines, antimicrobials β-defensins 2 and 3, and decreased bacterial plate counts. The data suggest that VIP modulates growth factor production in the infected cornea and that this in turn, contributes to better disease outcome.

Infection with Pseudomonas aeruginosa is often associated with keratitis, especially in users of extended-wear contact lenses,1–3 with disease outcome largely determined by the host inflammatory response. In fact, we propose that although an intense response may be needed to eradicate the bacteria, if left uncontrolled, it is often detrimental, favoring bystander tissue damage and in turn providing nutrients for bacterial growth and replication. Thus, tight regulation of inflammation and a balance between proinflammatory and antiinflammatory factors is requisite for disease resolution, including restoration of tissue homeostasis.4 Whether cells undergo necrosis and when they undergo apoptosis also may contribute to disease outcome. In this regard, previous studies5 have shown that apoptosis of polymorphonuclear leukocytes (PMNs) is delayed until 3 days p.i. in the corneas of susceptible C57BL/6 (B6) mice, whereas in resistant BALB/c mice, cells undergo apoptosis at 1 day p.i. The importance of earlier apoptosis in disease resolution was further confirmed in B6 mice by blocking interaction of the antiapoptotic neuropeptide substance P with its receptor (NK-1R), which resulted in earlier apoptosis and improved disease outcome. Apoptosis can be initiated by a variety of interactions, including the interaction between Fas and Fas Ligand (FasL), the well-documented death ligand and its receptor,6,7 leading to the subsequent activation of downstream caspases and the induction of cell lysis. It has been suggested that the Fas-FasL system may play an important role in the pathophysiology of infectious diseases.8 Evidence shows that P. aeruginosa infection induces lung epithelial cell apoptosis through the activation of endogenous Fas and FasL in vitro and in vivo, leading to improved disease outcomes in C3H mice.8 Others have reported that ExoS of P. aeruginosa triggers apoptosis in various cultured cell lines through clustering membrane Fas and activating the Fas-FasL pathway.9 Evidence has also suggested a direct regulatory role of the Fas-FasL system on local cytokine/chemokine production.10 For example, it was reported that C3H/HeJgld mice, which bear a nonfunctional mutation in FasL, showed significantly reduced Borrelia burgdorferi–specific cytokine response and reduced severity of arthritis.10 However, whether Fas-FasL interaction functions in P. aeruginosa–induced keratitis and has a similar impact on disease outcome remain to be determined. Our studies provide evidence that compared with WT mice, FasL−/− mice have a delayed onset of apoptosis and an exacerbated production of proinflammatory cytokines/chemokines and nitric oxide (NO) after P. aeruginosa corneal challenge, subsequently leading to worsening of disease. We also document in vitro that LPS-stimulated Mφ from FasL−/− mice compared with WT BALB/c mice showed decreased apoptosis, increased production of TNF-α, MIP-2, and IL-1β, and decreased production of IL-10, consistent with our in vivo findings.

Purpose Studies have shown that blocking substance P (SP) binding to neurokinin 1 receptor with spantide I prevents Pseudomonas aeruginosa-induced corneal perforation in susceptible C57BL/6 mice. This study tested the effect of SP injection on the resistance response (cornea heals) of BALB/c mice. Methods The day before infection, mice were injected intraperitoneally with SP or PBS. Disease was graded by clinical score, slit lamp, plate count, real-time RT-PCR, and ELISA assays, and polymorphonuclear neutrophils (PMNs) were quantitated using a myeloperoxidase assay. In additional experiments, BALB/c mice were injected intraperitoneally with vasoactive intestinal peptide (VIP) antagonist and similarly analyzed. Results Mice injected with SP exhibited worsened disease on days 1 to 7 after infection compared with controls. SP injection resulted in elevated PMN levels and viable bacterial counts in the cornea 3 and 5 days after infection. mRNA expression for NFkappaB and type 1 cytokines (e.g., IFN-gamma), as well as for TNF-alpha, MIP-2, IL-18, IL-6, and IL-1beta, were significantly elevated, whereas VIP and cytokines TGF-beta and IL-10 were significantly reduced. Differences in mRNA expression were selectively confirmed at the protein level by ELISA for NFkappaB, IL-1beta, and IL-10. VIP antagonist treatment also resulted in exacerbated disease scores, elevated proinflammatory mediators, and reduced anti-inflammatory mediators. Conclusions These data provide evidence that the neuropeptide SP, among its broad systemic effects, is a potent neuroimmunoregulator that promotes susceptibility in the resistant BALB/c mouse by overcoming the anti-inflammatory effects of VIP and IL-10 and that a balance between SP and VIP levels may be critical in disease resolution.

Purpose The purpose of this study was to determine the role of matrix metalloproteinases (MMP) in Pseudomonas aeruginosa keratitis. Methods Gene array and selective real-time PCR examined MMP expression in the cornea of susceptible (C57BL/6, B6) versus resistant (BALB/c) mice before and after infection; zymography tested enzyme activity for MMP-2 and -9. Clinical score, Langerhans cell (LC), and Neutrophil (PMN) quantitation were done in recombinant (r) MMP-9, antibody neutralized, and MMP-9(-/-) mice. The chemotactic potential of MMP-9 was tested in a Boyden chamber assay; light and transmission microscopy and immunostaining for collagen IV and MMP-9 were used to examine the effects and the source of MMP-9 after infection. ELISA was used to assess IL-1beta and MIP-2 levels. Results Gene array (confirmed by PCR) revealed sixfold more MMP-9, and zymography showed greater enzyme activity in the infected cornea of B6 over BALB/c mice. rMMP-9 injection of BALB/c mice enhanced, whereas MMP-9 antibody neutralization in B6 mice and its absence in MMP-9(-/-) mice decreased corneal disease. MMP-9(-/-) and antibody neutralized mice had fewer LCs in cornea; rMMP-9-treated mice had more. A myeloperoxidase (MPO) assay showed a similar pattern for PMN. MMP-9 was not chemotactic for LC or PMN. The basement membrane was more intact in MMP-9(-/-) over wild-type infected mice and correlated with staining for collagen IV; PMN was a source of MMP-9. IL-1beta and MIP-2 were increased in rMMP-9 but decreased in MMP-9 antibody neutralized and MMP-9(-/-) over control groups. Conclusions MMP-9 regulates immune function in cornea by proteolysis, potentiating P. aeruginosa keratitis by degrading collagen IV and upregulating chemotactic cytokines/chemokines IL-1beta and MIP-2.