Sharon A. McClellan

IL-33 Shifts Macrophage Polarization, Promoting Resistance against Pseudomonas aeruginosa Keratitis

PURPOSE To determine the role of IL-33 in resistance to Pseudomonas aeruginosa keratitis. METHODS Corneal IL-33 mRNA and protein levels were tested in susceptible C57BL/6 (B6) and resistant BALB/c mice. B6 mice were injected with recombinant mouse IL-33 (rmIL-33) and disease severity, bacterial load, polymorphonuclear neutrophils (PMN) infiltrate, gene expression of inflammatory, and T-helper (Th)1/Th2 cytokines were tested by RT-PCR. IL-33 signaling and macrophage (Mvarphi) polarization also were examined. RESULTS IL-33 mRNA and protein were expressed constitutively in the normal corneas of both groups and were significantly elevated at 1 to 5 days after infection in BALB/c over B6 mice. rmIL-33-treated B6 mice showed less severe disease than did PBS controls and exhibited decreased bacterial load, PMN infiltrate, and corneal mRNA levels for IL-1beta, MIP-2, and TNF-alpha. Th2-type cytokines (IL-4, -5, -10) also were significantly upregulated, and protein levels for TNF-alpha and IL-10 confirmed the mRNA data. To further investigate IL-33 in corneal inflammation, it was overexpressed in Mvarphi (RAW264.7 cells). This significantly increased IL-5 and IL-10, while it decreased IFN-gamma and other pro-inflammatory cytokines. The role of the Mvarphi was further tested in infected rmIL-33 compared with PBS-injected mice. Immunostaining showed that rmIL-33 injection shifted Mvarphi polarization from NO synthase 2 to arginase production. Furthermore, peritoneally elicited cells (B6 mice) treated with lipopolysaccharide and rmIL-33 exhibited elevated ST2 levels and a shift from IL-12 to IL-10 mRNA production. CONCLUSIONS These data provide evidence that IL-33 promotes a Th2-type immune response and reduces inflammation by polarizing the Mvarphi production of anti-inflammatory mediators in the cornea.

Macrophages Restrict Pseudomonas aeruginosa Growth, Regulate Polymorphonuclear Neutrophil Influx, and Balance Pro- and Anti-Inflammatory Cytokines in BALB/c Mice

The role of macrophages in Pseudomonas aeruginosa corneal infection in susceptible (cornea perforates), C57BL/6 (B6) vs resistant (cornea heals), BALB/c mice was tested by depleting macrophages using subconjunctival injections of clodronate-containing liposomes before corneal infection. Both groups of inbred mice treated with clodronate-liposomes compared with PBS-liposomes (controls) exhibited more severe disease. In B6 mice, the cornea perforated and the eye became extremely shrunken, whereas in BALB/c mice, the cornea perforated rather than healed. The myeloperoxidase assay detected significantly more PMN in the cornea of both groups of mice treated with clodronate-liposomes vs PBS-liposomes. In independent experiments, ELISA analysis showed that protein levels for IL-1β, macrophage-inflammatory protein 2, and macrophage-inflammatory protein 1α, all regulators of PMN chemotaxis, also were elevated in both groups of mice treated with clodronate-liposomes. Bacterial plate counts in B6 mice treated with clodronate-liposomes were unchanged at 3 days and were higher in control-treated mice at 5 days postinfection (p.i.), whereas in BALB/c mice, bacterial load was significantly elevated in the cornea of mice treated with clodronate-liposomes at both 3 and 5 days p.i. mRNA expression levels for pro (IFN-γ and TNF-α)- and anti (IL-4 and IL-10)-inflammatory cytokines also were determined in BALB/c mice treated with clodronate-liposomes vs control-treated mice. Expression levels for IFN-γ were significantly elevated in mice treated with clodronate-liposomes at 3 and 5 days p.i., while IL-10 levels (mRNA and protein) were reduced. These data provide evidence that macrophages control resistance to P. aeruginosa corneal infection through regulation of PMN number, bacterial killing and balancing pro- and anti-inflammatory cytokine levels.

β-Defensin-2 Promotes Resistance against Infection with P. aeruginosa

Corneal infection with Pseudomonas aeruginosa results in corneal perforation in susceptible C57BL/6 (B6) mice, but not in resistant BALB/c mice. To explore the role of two important defensins, murine β-defensin-1 (mBD1) and mBD2, in the ocular immune defense system, their mRNA and protein expression levels were tested by real-time RT-PCR and Western blot, respectively. mRNA, protein, and immunostaining data demonstrated that both mBD1 and mBD2 were constitutively expressed in normal BALB/c and B6 corneas, and they were disparately up-regulated in BALB/c (more) vs B6 (less) corneas after infection. To determine whether either defensin played a role in host resistance, BALB/c mice were treated with either mBD1 or mBD2 small interfering RNA by subconjunctival injection together with topical application. Increased corneal opacity and worsened disease were displayed after knockdown of mBD2 but not of mBD1. mBD2 silencing also increased bacterial counts and polymorphonuclear neutrophil infiltration in BALB/c corneas. Real-time RT-PCR data further demonstrated that mBD2, not mBD1, differentially modulated mRNA expression of proinflammatory cytokines/molecules such as IFN-γ, MIP-2, IL-1β, TNF-α, IL-6, and inducible NO synthase; TLR signaling molecules, including TLR2, TLR4, TLR9, and MyD88; and the transcription factor NF-κB. Additionally, in vivo studies indicated that mBD2 silencing enhanced corneal nitrite levels and NF-κB activation. Collectively, the data provide evidence that mBD2, but not mBD1, is required for host resistance against P. aeruginosa-induced corneal infection.

β-Defensins 2 and 3 together promote resistance to Pseudomonas aeruginosa keratitis

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.

IL-18 Contributes to Host Resistance Against Infection with Pseudomonas aeruginosa Through Induction of IFN-γ Production

Pseudomonas aeruginosa keratitis destroys the cornea in susceptible (B6), but not resistant (BALB/c) mice. To determine mechanisms mediating resistance, the role of IFN-γ, IL-12, and IL-18 was tested in BALB/c mice. RT-PCR analysis detected IFN-γ mRNA expression levels in cornea that were significantly increased at 1–7 days postinfection. IL-18 mRNA was detected constitutively in cornea and, at 1–7 days postinfection, levels were elevated significantly, while no IL-12 mRNA was similarly detected. To test whether IL-18 contributed to IFN-γ production, mice were treated with anti-IL-18 mAb. Treatment decreased corneal IFN-γ mRNA levels, and bacterial load and disease increased/worsened, compared with IgG-treated mice. To stringently examine the role of IFN-γ in bacterial killing, knockout (−/−) vs wild-type (wt) mice also were tested. All corneas perforated, and bacterial load was increased significantly in −/− vs wt mice. Because disease severity was increased in IFN-γ−/− vs IL-18-neutralized mice, and since IL-18 also induces production of TNF, we tested for TNF-α in both groups. ELISA analysis demonstrated significantly elevated corneal TNF-α protein levels in IFN-γ−/− vs wt mice after infection. In contrast, RT-PCR analysis of IL-18-neutralized vs IgG-treated infected mice revealed decreased corneal TNF-α mRNA expression. Next, to resolve whether TNF was required for bacterial killing, TNF-α was neutralized in BALB/c mice. No difference in corneal bacterial load was detected in neutralized vs IgG-treated mice. These data provide evidence that IL-18 contributes to the resistance response by induction of IFN-γ and that IFN-γ is required for bacterial killing.

HIF-1α Is Essential for Effective PMN Bacterial Killing, Antimicrobial Peptide Production and Apoptosis in Pseudomonas aeruginosa Keratitis

Hypoxia-inducible factor (HIF)-1α, is a transcription factor that controls energy metabolism and angiogenesis under hypoxic conditions, and a potent regulator of innate immunity. The studies described herein examined the role of HIF-1α in disease resolution in BALB/c (resistant, cornea heals) mice after ocular infection with Pseudomonas (P.) aeruginosa. Furthermore, the current studies focused on the neutrophil (PMN), the predominant cell infiltrate in keratitis. Using both siRNA and an antagonist (17-DMAG), the role of HIF-1α was assessed in P. aeruginosa-infected BALB/c mice. Clinical score and slit lamp photography indicated HIF-1α inhibition exacerbated disease and corneal destruction. Real time RT-PCR, immunohistochemistry, ELISA, Greiss and MPO assays, bacterial load, intracellular killing, phagocytosis and apoptosis assays further tested the regulatory role of HIF-1α. Despite increased pro-inflammatory cytokine expression and increased MPO levels after knocking down HIF-1α expression, in vivo studies revealed a decrease in NO production and higher bacterial load. In vitro studies using PMN provided evidence that although inhibition of HIF-1α did not affect phagocytosis, both bacterial killing and apoptosis were significantly affected, as was production of antimicrobial peptides. Overall, data provide evidence that inhibition of HIF-1α converts a normally resistant disease response to susceptible (corneal thinning and perforation) after induction of bacterial keratitis. Although this inhibition does not appear to affect PMN transmigration or phagocytosis, both in vivo and in vitro approaches indicate that the transcriptional factor is essential for effective bacterial killing, apoptosis and antimicrobial peptide production.

β-Defensins 2 and 3 Together Promote Resistance to P. aeruginosa Keratitis

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.

NKT Cells Are Critical to Initiate an Inflammatory Response after Pseudomonas aeruginosa Ocular Infection in Susceptible Mice

CD4+ T cells produce IFN-γ contributing to corneal perforation in C57BL/6 (B6) mice after Pseudomonas aeruginosa infection. To determine the role of NK and NKT cells, infected corneas of B6 mice were dual immunolabeled. Initially, more NKT than NK cells were detected, but as disease progressed, NK cells increased, while NKT cells decreased. Therefore, B6 mice were depleted of NK/NKT cells with anti-asialo GM1 or anti-NK1.1 Ab. Either treatment accelerated time to perforation, increased bacterial load and polymorphonuclear neutrophils, but decreased IFN-γ and IL-12p40 mRNA expression vs controls. Next, RAG-1 knockout (−/−; no T/NKT cells), B6.TCR Jα281−/− (NKT cell deficient), α-galactosylceramide (αGalCer) (anergized NKT cells) injected and IL-12p40−/− vs B6 controls were tested. IFN-γ mRNA was undetectable in RAG-1−/−- and αGalCer-treated mice at 5 h and was significantly reduced vs controls at 1 day postinfection. It also was reduced significantly in B6.TCR Jα281−/−, αGalCer-treated, and IL-12p40−/− (activated CD4+ T cells also reduced) vs control mice at 5 days postinfection. In vitro studies tested whether endotoxin (LPS) stimulated Langerhans cells and macrophages (Mφ; from B6 mice) provided signals to activate NKT cells. LPS up-regulated mRNA expression for IL-12p40, costimulatory molecules CD80 and CD86, NF-κB, and CD1d, and addition of rIFN-γ potentiated Mφ CD1d levels. Together, these data suggest that Langerhans cell/Mφ recognition of microbial LPS regulates IL-12p40 (and CD1d) driven IFN-γ production by NKT cells, that IFN-γ is required to optimally activate NK cells to produce IFN-γ, and that depletion of both NKT/NK cells results in earlier corneal perforation.

Mammalian Target of Rapamycin Regulates IL-10 and Resistance to Pseudomonas aeruginosa Corneal Infection

IL-10 is important in the resistance response of BALB/c mice to experimental Pseudomonas aeruginosa corneal infection. However, the cellular mechanisms by which this anti-inflammatory cytokine is regulated remain unknown. Because the mammalian target of rapamycin (mTOR) regulates IL-10 in other disease models, the present study tested its role in bacterial keratitis. After infection, corneas of rapamycin versus control-treated BALB/c mice showed worsened disease, and real-time RT-PCR confirmed that mTOR mRNA levels were significantly decreased. Rapamycin treatment also increased clinical score, polymorphonuclear neutrophil (PMN) infiltration (determined by myeloperoxidase assay), and bacterial load, but it diminished PMN bactericidal activity. Inhibition of mTOR also led to elevated mRNA and protein levels of IL-12p40, matrix metalloproteinase 9, and inducible NO synthase, whereas mRNA and protein levels of IL-10, its regulator/effector STAT-3, and suppressor of cytokine signaling 3 (a proinflammatory cytokine regulator) were decreased. Furthermore, mTOR inhibition reduced levels of proapoptotic caspase-3 and increased levels of B cell lymphoma-2 (antiapoptotic), indicative of delayed apoptosis. mTOR inhibition also altered genes related to TLR signaling, including elevation of TLR4, TLR5, and IL-1R1, with decreases in IL-1R-associated kinase 1 and an inhibitor of NF-κB, NF-κB inhibitor–like 1. Rapamycin treatment also increased levels of IFN-γ and CCAAT/enhancer binding protein, β, a gene that regulates expression of preprotachykinin-A (the precursor of substance P). Collectively, these data, as well as a rescue experiment using rIL-10 together with rapamycin, which decreased PMN in cornea, provide concrete evidence that mTOR regulates IL-10 in P. aeruginosa–induced bacterial keratitis and is critical to balancing pro- and anti-inflammatory events, resulting in better disease outcome.

Vasoactive Intestinal Peptide Downregulates Proinflammatory TLRs While Upregulating Anti-Inflammatory TLRs in the Infected Cornea

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.