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There is increasing interest in TLRs in the pathogenesis of eye and skin disease.
The skin is an organ that contains many specialized cells and structures. The skin functions as a barrier that protects us from a hostile environment. It is also very involved in maintaining the proper temperature for the body to function well. It gathers sensory information from the environment, and plays an active role in the immune system protecting us from disease. It is composed of 3 layers - the epidermis, dermis, and subcutaneous tissue. Within these layers there is further differentiation. An important cell type in the skin is the Langerhan cell, which is an immature DC that migrates to the local lymph node upon stimulation.
The ocular surface is constantly exposed to a wide array of microorganisms. The ability of the cornea to recognize pathogens as foreign and eliminate them is critical to retain its transparency, hence preservation of sight. In the eye, as in other parts of the body, the early response against invading pathogens is provided by innate immunity. The eye is said to be "immune privileged", without immune privilege, even minor episodes of inflammation in the eye could damage the cornea or retina, causing impaired vision or even blindness. Immune privilege prevents that bystander damage. It also is likely that immune privilege is one reason the cornea can be transplanted without the need for potent anti-rejection drugs. Fas/FasL interactions are important in the maintenance of this state. However, this system is not perfect and immune mediated diseases of eye. For example, uveitis is inflammation inside the eye, specifically affecting one or more of the three parts of the eye that make up the uvea: the iris (the colored part of the eye), the ciliary body (behind the iris, responsible for manufacturing the fluid inside the eye), and the choroid (the vascular lining tissue underneath the retina). After diabetes and macular degeneration, it is the third most common cause of blindness in the US.
TLR Expression & Effector Function in the Skin
One study has demonstrated that normal keratinocytes express TLR1, 2, and 5 (Baker et al, 2003). This study utilized punch biopsies from regions of normal skin in psoriasis patients and normal breast skin obtained from donors with no known skin diseases. Antibody staining of the biopsies demonstrated cytoplasmic TLR1 and TLR2 expression throughout the epidermis with TLR2 staining most strongly on basal keratinocytes. The basal layer also demonstrated TLR5 staining. Thus, it appears that keratinocytes in different layers of the epidermis may express different TLR; as keratinocytes mature as they progress from the basal layer to the surface of the skin, their patterns of TLR expression may also change. Although not detected by Baker et al., other studies report expression of TLR4 on keratinocytes. Pivarcsi et al (2003) demonstrated TLR2 and TLR4 mRNA and protein expression in cultured human epidermal keratinocytes obtained from the skin of healthy individuals. Also, antibody staining of skin sections demonstrated the presence of TLR2 and TLR4 throughout the epidermis.
Most recently, Mempel et al (2003) reported that cultured primary human keratinocytes expressed TLR1,2,3,5, and 9, but TLR4,6,7, and 8 were undetectable. These conflicting reports make it unclear if keratinocytes constitutively express TLR4.
Mempel et al found stimulation of keratinocytes with Staphylococcus aureus caused translocation of NFkB and subsequent increased production of IL-8 and iNOS. This inflammatory response was found to be TLR2 dependent (Mempel et al, 2003) TLR-activated keratinocytes are also capable of modulating the host's adaptive immune response. Lebre et al (2003) demonstrated that supernatants of TLR-stimulated keratinocytes induced the maturation of human monocytederived immature DC. These now-mature DC were found to promote Th1 immune responses from naÄ±ve T cells.
In contrast to DC, LC have been shown to respond differently to microbial TLR ligands. For example, LPS is capable of inducing the maturation of DC but not LC. Moreover, stimulation with LPS leads to the upregulation of CD80, CD86, and HLA-DR on DC but not LC. This relative unresponsiveness of LC has been linked to lower levels of TLR expression on the LC (Takeuchi et al, 2003). More specifically, this group could not detect mRNA for TLR4 in LC, and the amount of mRNA encoding TLR2 found in LC was demonstrably less than that found in DC. In addition, stimulation with ligands for TLR2, 4, and 9 matured splenic DC but not LC.
TLRs in Dermatological disease
Although there has been increased interest recently on the role of TLRs in the pathogenesis of skin and eye disease, not much is known about their role in these diseases. However, TLR stimulation influences T cell development, and by blocking this pathway in diseases such as psoriasis could be beneficial. MyD88 deficient mice developed a profound defect in the antigen-specific Th1 but not Th2 responses, suggesting that TLR signals play an influential role in the immune balance toward to Th1 but not Th2 response (Schnare et al., 2001). CD4+ Th1 cells are critical in the pathogenesis of psoriasis. In fully developed psoriatic skin lesions there is a mixture of innate immune cells (neutrophils, dendritic APCs andNKTcells), adaptive immune (T) cells, and an inflammatory infiltrate. Both CD4+ and CD8+ T lymphocytes are present, with the CD41 T lymphocytes being present mostly in the dermis. Table 1 shows the types of T cells that play a role in psoriasis. Activation of TLR2 on dendritic cells (DCs) selectively induced IL-12 but not IL-10 (Thoma-Uszynski et al., 2000).
DCs are critical in the development of T cell responses. Human blood contains at least two types of DCs, CD11c+ and plasmacytoid DC (PDC). CD11c+DC selectively expresses most TLRs but not TLR9. In contrast, PDCs strongly express TLR9 but lack TLR3, 4 and 8 (Kadowaki et al., 2001, Jarossay et al., 2001). In accordance with their TLR expression profiles, CD11c+DC do not respond to the TLR9 ligand CpG DNA but do respond to TLR2 ligands to produce TNF-Î±, but not IL-12 (Boonstra et al., 2003). In contrast, PDC failed to respond to lipoteichoic acid, poly I:C and LPS, but responded to CpG and induced strong Th1 differentiation (Kadowaki et al., 2001, Jarossay et al., 2001). This selective expression of TLRs and the differential response to microbial products by DC subsets may give rise to distinct guiding of Th cell development.
Activation of TLRs results in secretion of proinflammatory cytokines and induction of direct antimicrobial mechanisms. However, TLR activation can induce cellular apoptosis and the robust inflammation often leads to concomitant tissue injury (Krutzik et al., 2001), for example nerve damage in leprosy (Oliveira et al., 2003), myocardial ischemia/reperfusion injury, the manifestations of septic shock, and the pathogenesis of inflammatory acne (Kim et al., 2002).
Psoriasis is a common skin disease that is mediated by Th1 cells. The psoriatic plaque is characterized by the presence of Th1-type cytokines (IFN-Î³, IL-2, and TNF-Î±), but not Th2-type cytokines such as IL-4, IL-5, or IL-10 (Austin et al., 1999). In addition to T-lymphocyte derived cytokines, the numerous APCs that infiltrate into skin lesions also contribute to the localized inflammatory infiltrate, which includes IL-18, IL-23, and TNF-Î±. Both IL-18 and IL-23 are inducers of IFN-Î³ production by Th1 lymphocytes. It is IL-23 and not IL-12 that is dominant in psoriatic plaques (Lee et al., 2004).
TNF-Î± is a cytokine that deserves special attention in psoriasis. Chronic overproduction of TNF-Î± in the skin, joint, and bowel wall has been associated with chronic inflammatory conditions such as psoriasis, psoriatic arthritis, rheumatoid arthritis, and inflammatory bowel disease. The important role of inflammation related to TNF-Î± biologic activity in psoriasis has been demonstrated by the efficacy of TNFÎ± targeting agents (etanercept, infliximab, and adalimumab) in the treatment of psoriasis. Blockade of TNF-Î± by blocking TLR2 signaling would be a novel way of treating Th1 mediated skin diseases. An immunohistochemical study by Baker et al., (2003) showed that TLR1 and 2 were upregulated in psoriatic keratinocytes in the suprabasal layer.
Acne vulgaris is a very common disorder that affects 17 million people in the US alone (Kim et al., 2002). The pathogenesis of acne is multifactoral but involves an immune mechanism. The causative agent is P.acnes and it contributes to the inflammatory nature of the disease by inducing monocytes to secrete IL-1Î², TNF-Î± and IL-8. IL-12p40, a subunit common to both IL-12 and IL-23, promoter activity was also mediated by TLR2. Blockade of TLR2 signaling using all-trans retinoids, a treatment for acne, results in significant downregulation of IL-12 and TNF-Î± (Liu et al., 2005) and could be a novel treatment for acne.
Leprosy, a disease caused by infection with the organism mycobaterium varies widely in its clinical presentation, which can be correlated with the type of immune response the host has launched against M. leprae. The tuberculoid form of the disease is characterized by localized infection, granulomatous lesions, and the expression of type 1 cytokines that promote cell-mediated immunity. On the other end of the spectrum is the lepromatous form of the disease that is characterized by disseminated infection, disfiguring nodular lesions, and the expression of type 2 cytokines that promote a humoral immune response. A recent study demonstrated that heterodimers of TLR2/1 were activated by killed M. leprae (Krutzik et al, 2003). Since earlier studies indicate TLR2/1 heterodimers recognize triacylated lipoproteins, the genome of M. leprae was scanned to identify putative lipoproteins. Further experiments revealed that two lipoproteins, identified as 19 and 33 kDa, were capable of both monocyte and DC activation. Schwann cells found in leprosy skin lesions were shown to express TLR2, and apoptotic Schwann cells were demonstrated within the lesions. Thus, lipopeptides from M. leprae may promote TLR-induced apoptosis in Schwann cells. In light of these studies, the nerve injury observed in leprosy patients appears to be a consequence of the innate immune response to M. leprae.
Inflammatory Ocular Diseases
Inflammatory eye diseases can affect any part of the eye from the ocular surface to the retina, the optic nerve, and other orbital structures. The consequences of inflammation in the eye, whether appropriate (as in the case of immune response to infective threats) or inappropriate (as in the case of autoimmune or allergic responses), may be sight threatening. Consequently, the eye normally exhibits ''immune privilege'' to avoid the potential sight destroying consequences of ocular inflammation. Ocular immune privilege involves certain anatomical, cellular, and soluble factors, such as the blood-ocular barrier and immunosuppressive factors of the aqueous humour. TLRs have been implicated in the pathogenesis of chronic inflammatory diseases such as inflammatory bowel disease, rheumatoid arthritis, psoriasis, and multiple sclerosis (Takeda et al., 2003). Interestingly, these diseases are not uncommonly associated with various inflammatory eye diseases, such as uveitis and scleritis.
Potential role of TLRs in Ocular Disease
The cornea forms an avascular and transparent window at the ocular surface that functions to maintain a clear visual axis as well as protect against the elements of the harsh external environment, including microbial threats. Analogous to the expression of TLRs found at other epithelial surfaces (Takeda et al, 2003; Backhed et al., 2003). it might be expected that the cornea would also be similarly endowed with a pattern of TLRs to defend the integrity of the ocular surface. However, the cornea is also critical in the maintenance of visual function, and activation of such TLRs in the cornea could result in inflammation, which might compromise corneal transparency and vision. TLR activation may be inappropriate and self destructive if it is in response to the non-pathogenic,
normal commensal flora of the ocular surface. The reported expression of functional TLR2 by cultured corneal epithelial cells, which produced pro-inflammatory cytokines, chemokines, and antimicrobial peptide in response to peptidoglycan of Staphylococcus aureus, may have a role in the pathogenesis of Gram positive bacterial keratitis (Kumar et al., 2004). However, it has since been shown that peptidoglycan is not a TLR2 ligand. In contrast with these studies, Ueta et al (2004) found that human corneal epithelial cells expressed TLR2 and TLR4 intracellularly but not at the cell surface.
TLR4 has been also implicated to have a critical role in the inflammatory response associated with the pathogenesis of ocular onchocerciasis (river blindness), (Saint Andre et al., 2002). Onchocerca volvulus is a parasitic nematode transmitted by the Simulium blackfly that causes corneal inflammation, which can lead to blindness. Recent studies using TLR2 KO, TLR9 KO, and MyD88 KO mice have further supported the in vivo functional importance of the activation of TLR/MyD88 pathway in the development of corneal inflammation (Johnson et al., 2005). These studies examined the development of keratitis after stimulation of the cornea of TLR KO mice that had received a superficial corneal abrasion with the relevant TLR ligands. For example, CpG DNA (TLR9 ligand) was able to induce keratitis in wild type mice but not in the TLR9 KO mice. Endotoxin induced keratitis (via TLR4) could be normally induced in these TLR9 KO mice. Furthermore, the ligands for TLR2, TLR-4, or TLR-9 were not able to induce keratitis in mice that lacked the common adaptor protein, MyD88, required for TLR intracellular signalling. Although these models are quite crude, they demonstrate that the murine cornea has functional TLR2, TLR-4, and TLR-9, and that activation of these by their respective PAMPs induces the development of keratitis, in a MyD88 dependent manner, by the secretion of chemokines and the recruitment of neutrophils into the corneal stroma (Johnson et al., 2005).
Uveitis is a heterogeneous group of diseases characterised by acute, recurrent, or chronic inflammation of the uvea, the middle vascular coat of the eye. It is the most common cause of intraocular inflammatory disease and an important cause of visual impairment in most populations (Chang et al., 2002). Uveitis may be infectious in aetiology or non-infectious, predominantly immune mediated. In endotoxin induced uveitis (EIU), a well established animal model for human AAU, LPS of Gram negative bacterial cell wall, when injected at sites remote from the eye induces an AAU without significantly affecting other tissues (Rosenbaum et al., 1980, Chang et al., 2005). A highly selective and specific pattern of TLR4 protein expression was observed in the human uvea, with the epithelial and endothelial cells of the normal human iris and ciliary body not expressing TLR4 and MD-2 proteins. IThis is likely due to the unique immunologically privileged nature of the eye. Although the human iris endothelium does not express TLR4 protein in vivo, cultured human iris endothelial cells were found to express TLR4 mRNA and respond to LPS stimulation with the production of pro-inflammatory cytokines (Brito et al., 2004). This may have implications for understanding the apparent susceptibility of the anterior uvea to the breakdown of the blood-aqueous barrier and development of uveitis. Importantly, Brito et al showed that TLR4 in the human uvea can be functional, by demonstrating TLR4 dependent proinflammatory cytokine production in response to in response to in vitro LPS stimulation of cultured uveal tissue explants.
There appears to be a particular predilection for the involvement of the retina by various infectious agents, such as viruses (CMV and HSV retinitis) and the obligate intracellular protozoan, Toxoplasma gondii, the commonest cause of posterior uveitis (Chang et al., 2002). Retinal pigment epithelium (RPE) is a monolayer of cells strategically located between the neurosensory retina and the vascular choroid, forming part of the blood-retina barrier, and have been implicated to have a role in the immunopathogenesis of uveoretinitis. Cultured human RPE cells expressed the genes for TLRs 1- 7, TLR-9, and TLR-10, with the highest expression level found for TLR3. TLR3 stimulation of RPE cells induced the secretion of various cytokines, chemokines, and adhesion molecule (Kumar et al., 2004). Thus, TLR3 mediated signalling, triggered by dsRNA of virus replication, may have a protective role in viral infections of the retina. RPE cells appear to be one of the principal cellular targets for infectious agents, such as CMV and Toxoplasma gondii (Detrick et al., 1996). TLRs have been implicated in innate immune recognition of T gondii. In particular, TLR2 appears to have an important role in the innate immune response to T gondii, although additional TLRs and ligands have also been implicated (Scanga et al., 2002, Mun et al., 2003).
There appears to be intense interest in TLRs in both eye and skin disease. It has been shown that TLRs are expressed in eye tissue but as yet their role has not been elucidated in non-microbial based inflammatory diseases. It has yet to be formally shown that TLR agonists can ameliorate inflammatory disease, it is however quite likely that inhibition of NF-ÎºB would be beneficial in these diseases. As such, there is potential in inhibition of TLR signalling in the treatment of inflammatory ocular diseases.
With regard to inflammatory skin conditions, there is evidence that inhibition of TLR signalling, particularly TLR2, is beneficial in the treatment of diseases such as psoriasis, acne and leprosy. TLR2 has been shown to be upregulated in psoriatic lesions, and the inflammatory cytokine induction in acne is mediated by TLR2. There are models of psoriasis, some of which involve transplanting psoriatic skin onto the backs of immunodeficient mice, that could be utilised in the OPN301 project.
Rhino mice are used to investigate the effects of comedolytic agents, i.e. those that prevent acne. Rhino mice have normal-appearing skin and hair at birth. At the end of the first hair cycle in hrrh/hrrh mice, the dermal papilla fails to follow the contracting follicle and becomes isolated in the reticular dermis and hypodermis. These do not become reassociated to start the hair cycle over again. By three weeks of age, small lumen develop that enlarge with age forming deep dermal cysts. Sebaceous glands undergo hypertrophy at 30 days of age and atrophy after 1 year of age. These mice could potentially be used if the role of TLR2 was to be investigated in acne.