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

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.