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A group of proteases in the endosomal/lysosomal proteolytic system have been designated as cathepsins, which is derived from the Greek term meaning "to digest". Considering that cathepsins can irreversibly cleave peptide bonds, the primary function of cathepsins has been believed to be their "disintegration action". However, there is increasing evidence that cathepsins can also exert "modulator actions" by which substrates are activated after limited cleavage. There is substantial evidence that cathepsin B (EC <IP_ADDRESS>), a typical cysteine lysosomal protease, is markedly upregulated in activated microglia that accumulate in pathological sites of the brain. Beyond its bulk proteolysis in the endosomal/lysosomal system, cathepsin B can be secreted from activated microglia in its mature form to induce neuronal apoptosis and degrade A peptides that accumulate in the brain. Furthermore, cathepsin B is also leaked into the cytosol, where it plays an essential role in the inflammatory response initiated by activated microglia in the brain.

Recently, the leakage of cathepsin B from the lysosomes has been suggested to trigger the activation of the NOD-like receptor (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome in microglia/macrophages after phagocytosis of various molecules including fibrillar A42 and silica crystals. After activation, the NLRP3 inflammasome can mediate pro-caspase-1 activation to promote the processing and secretion of proinflammatory cytokines, such as interleukin-1 (IL-1) and IL-18. However, the precise role of leaked cathepsin B in the activation of the NLRP3 inflammasome remains to be determined. Furthermore, there is still evidence suggesting that cathepsin B is associated with the maturation of pro-IL-1 in the endosomal/lysosomal system, because cathepsin B can effectively cleave pro-caspase-1 in a cell-free system only at an acidic pH. I herein review our current understanding of the mechanism and roles of cathepsin B in the processing and secretion of IL-1 and IL-18. Further, i also discuss a possible involvement of cathepsin B in the induction of persistent pain.

Involvement of Microglial Cathepsin B in Pro-Interleukin-1 Processing and Persistent Pain 267

have recently been proposed to account for perturbations in phagocytic processes that activate the NLRP3 inflammasome. According to the lysosomal rupture model, phagocytosis of fibrillar A42 or silica crystals by LPS-primed microglia/macrophages causes phagosomal destabilization and lysosomal rupture. The subsequent secretion of cathepsin B into the cytoplasm triggers the activation of the NLRP3 inflammasome directly or indirectly, leading to the production and secretion of mature IL-1(Hornung et al., 2008;

CGA: chromogranin A; IAPP: islet amyloid peptide; MSU: monosodium urate crystal

pathways, which are associated with various diseases (Table 1).

This model is supported by observations that a specific inhibitor of cathepsin B, CA074Me, significantly inhibited the IL-1 secretion from LPS-primed microglia and macrophages after phagocytosis of fibrillar A and silica crystal, respectively (Hornung et al., 2008; Halle et al., 2008). Furthermore, the mean level of IL-1 secreted from cathepsin B-deficient macrophages following the phagocytosis of fibrillar Awas significantly lower than that from wild-type macrophages (Halle et al., 2008). Interestingly, CA074Me had no effect on the amount of IL-1 secreted from LPS-primed microglia following treatment with ATP (Table 1). Furthermore, a direct disruption of lysosomes by treatment with hypotonic media or L-leucyl-L-leucil methyl ester was shown to be sufficient for the activation of NLRP3 (Hornung et al., 2008). On the other hand, according to the ROS model, particulate activators of the NLRP3 inflammasome including asbestos fibers and silica crystals trigger the generation of short-lived ROS, and treatment with various ROS scavengers blocks the activation of the NLRP3 inflammasome in response to these particulate activators (Dostert et al., 2008) . Various danger signals stimulate phagocytes to induce various IL-1 production

More recently, however, reports opposing both of these models have appeared. Dostert et al. (2009) demonstrate that cathepsin B-deficient macrophages exhibited a normal NLRP3 inflammasome-dependent IL-1 production in response to silica crystals, urea crystals or aluminum salts, which raises questions about the specificity of CA074Me. Lysosomal rupture is associated with the release of numerous other enzymes, therefore CA074Me might inhibit other released proteases that acts as the essential signal to activate the NLRP3 inflammasome (Montaser et al., 2002). In fact, cathepsin L-deficient macrophages were also used to show that cholesterol crystals led to a diminished release of IL-1 in comparison with wild-type cells (Duewell et al., 2010). However, the dependence of cholesterol crystal-

Table 1. IL-1 production pathways and diseases

Halle et al., 2008).

### **2. Cathepsin B and neuronal death**

#### **2.1 Neuronal death induced by cathepsin B-secreted from microglia**

Microglia are known to release a number of soluble molecules that can influence neuronal signaling and survival. Kingham and Pocock (2001) focused on cathepsin B, which increased in the culture medium of microglia following stimulation with chromogranin A (CGA), a glycophosphoprotein secreted by degenerating neurons. They demonstrated that cathepsin B is a major causative factor for CGA-activated microglia-induced neuronal apoptosis using neutralizing anti-cathepsin B antibodies. Gan et al. (2004) found that freshly sonicated A42 did not cause neuronal death when added directly to neuron, but activated BV2 microglial cells to release toxic factors that caused significant neuronal death. To determine the toxic molecules secreted from A42-stimulated microglia, they conducted a large scale expression profiling analyses using filter-based cDNA arrays made from BV2 cDNA libraries enriched for A42-activated microglial genes. Cathepsin B was identified to be one of the 554 genes transcriptionally induced by freshly sonicated A42. Furthermore, specific inhibition of cathepsin B using either siRNA-mediated gene silencing or a specific cathepsin B inhibitor completely abolished the neurotoxicity mediated by A42-activated BV2 microglial cells, suggesting that cathepsin B plays a crucial role in neuronal death mediated by A-activated inflammatory responses. However, further studies will be needed to identify the mechanism of secretion of cathepsin B and its extracellular substrates.

#### **2.2 Involvement of intracellular cathepsin B in microglia-induced neuronal death**

Wendt et al. (2009) analyzed the neurotoxicity of conditioned medium from lipopolysaccharide (LPS)-activated microglia. Experiments with membrane-permeable and membrane-impermeable cathepsin B inhibitors suggested that blocking extracellular cathepsin B had no effect on LPS-stimulated microglia-mediated neuronal death. In contrast, intracellular cathepsin B may trigger the release of neurotoxic factors from activated microglia. In fact, it has been reported that cathepsin B is involved in the trafficking of tumor necrosis factor--containing vesicles to the plasma membrane of macrophages (Ha et al., 2008).

#### **3. Cathepsin B and A**

To determine the role of cathepsin B in the processing of amyloid precursor protein and A metabolism in vivo, Mueller-Steiner et al. (2006) crossed cathepsin B-deficient mice with transgenic mice that overexpressed human amyloid precursor protein (hAPP mice). Cathepsin B ablation in hAPP mice did not affect the levels of the C-terminal fragments of hAPP, suggesting that cathepsin B dose not significantly affect the processing of hAPP. In contrast, cathepsin B ablation significantly increased the plaque deposition and A42 levels in the hippocampaus. On the other hand, the injection of Lenti-cathepsin B into the hippocampus significantly reduced A deposition in aged hAPP mice. These observations strongly suggest that cathepsin B is secreted from microglia accumulated around the senile plaques, and is involved in the degradation of A42 and reduction of established plaques.

#### **4. Cathepsin B and inflammation**

#### **4.1 The "lysosomal rupture model" and "reactive oxygen species (ROS) model" of activation of the NLRP3 inflammasome**

Two different models, the "lysosomal rupture model" proposed by Latz's group (University of Bonn) and the "ROS model" proposed by Tschopp's group (University of Lausanne), have recently been proposed to account for perturbations in phagocytic processes that activate the NLRP3 inflammasome. According to the lysosomal rupture model, phagocytosis of fibrillar A42 or silica crystals by LPS-primed microglia/macrophages causes phagosomal destabilization and lysosomal rupture. The subsequent secretion of cathepsin B into the cytoplasm triggers the activation of the NLRP3 inflammasome directly or indirectly, leading to the production and secretion of mature IL-1(Hornung et al., 2008; Halle et al., 2008).

CGA: chromogranin A; IAPP: islet amyloid peptide; MSU: monosodium urate crystal

Table 1. IL-1 production pathways and diseases

Inflammation, Chronic Diseases and Cancer –

266 Cell and Molecular Biology, Immunology and Clinical Bases

Microglia are known to release a number of soluble molecules that can influence neuronal signaling and survival. Kingham and Pocock (2001) focused on cathepsin B, which increased in the culture medium of microglia following stimulation with chromogranin A (CGA), a glycophosphoprotein secreted by degenerating neurons. They demonstrated that cathepsin B is a major causative factor for CGA-activated microglia-induced neuronal apoptosis using neutralizing anti-cathepsin B antibodies. Gan et al. (2004) found that freshly sonicated A42 did not cause neuronal death when added directly to neuron, but activated BV2 microglial cells to release toxic factors that caused significant neuronal death. To determine the toxic molecules secreted from A42-stimulated microglia, they conducted a large scale expression profiling analyses using filter-based cDNA arrays made from BV2 cDNA libraries enriched for A42-activated microglial genes. Cathepsin B was identified to be one of the 554 genes transcriptionally induced by freshly sonicated A42. Furthermore, specific inhibition of cathepsin B using either siRNA-mediated gene silencing or a specific cathepsin B inhibitor completely abolished the neurotoxicity mediated by A42-activated BV2 microglial cells, suggesting that cathepsin B plays a crucial role in neuronal death mediated by A-activated inflammatory responses. However, further studies will be needed to identify the mechanism

**2.2 Involvement of intracellular cathepsin B in microglia-induced neuronal death**  Wendt et al. (2009) analyzed the neurotoxicity of conditioned medium from lipopolysaccharide (LPS)-activated microglia. Experiments with membrane-permeable and membrane-impermeable cathepsin B inhibitors suggested that blocking extracellular cathepsin B had no effect on LPS-stimulated microglia-mediated neuronal death. In contrast, intracellular cathepsin B may trigger the release of neurotoxic factors from activated microglia. In fact, it has been reported that cathepsin B is involved in the trafficking of tumor necrosis

factor--containing vesicles to the plasma membrane of macrophages (Ha et al., 2008).

To determine the role of cathepsin B in the processing of amyloid precursor protein and A metabolism in vivo, Mueller-Steiner et al. (2006) crossed cathepsin B-deficient mice with transgenic mice that overexpressed human amyloid precursor protein (hAPP mice). Cathepsin B ablation in hAPP mice did not affect the levels of the C-terminal fragments of hAPP, suggesting that cathepsin B dose not significantly affect the processing of hAPP. In contrast, cathepsin B ablation significantly increased the plaque deposition and A42 levels in the hippocampaus. On the other hand, the injection of Lenti-cathepsin B into the hippocampus significantly reduced A deposition in aged hAPP mice. These observations strongly suggest that cathepsin B is secreted from microglia accumulated around the senile plaques, and is involved in the degradation of A42 and reduction of established plaques.

**4.1 The "lysosomal rupture model" and "reactive oxygen species (ROS) model" of**

Two different models, the "lysosomal rupture model" proposed by Latz's group (University of Bonn) and the "ROS model" proposed by Tschopp's group (University of Lausanne),

**2.1 Neuronal death induced by cathepsin B-secreted from microglia**

of secretion of cathepsin B and its extracellular substrates.

**3. Cathepsin B and A**

**4. Cathepsin B and inflammation**

**activation of the NLRP3 inflammasome**

**2. Cathepsin B and neuronal death**

This model is supported by observations that a specific inhibitor of cathepsin B, CA074Me, significantly inhibited the IL-1 secretion from LPS-primed microglia and macrophages after phagocytosis of fibrillar A and silica crystal, respectively (Hornung et al., 2008; Halle et al., 2008). Furthermore, the mean level of IL-1 secreted from cathepsin B-deficient macrophages following the phagocytosis of fibrillar Awas significantly lower than that from wild-type macrophages (Halle et al., 2008). Interestingly, CA074Me had no effect on the amount of IL-1 secreted from LPS-primed microglia following treatment with ATP (Table 1). Furthermore, a direct disruption of lysosomes by treatment with hypotonic media or L-leucyl-L-leucil methyl ester was shown to be sufficient for the activation of NLRP3 (Hornung et al., 2008). On the other hand, according to the ROS model, particulate activators of the NLRP3 inflammasome including asbestos fibers and silica crystals trigger the generation of short-lived ROS, and treatment with various ROS scavengers blocks the activation of the NLRP3 inflammasome in response to these particulate activators (Dostert et al., 2008) . Various danger signals stimulate phagocytes to induce various IL-1 production pathways, which are associated with various diseases (Table 1).

More recently, however, reports opposing both of these models have appeared. Dostert et al. (2009) demonstrate that cathepsin B-deficient macrophages exhibited a normal NLRP3 inflammasome-dependent IL-1 production in response to silica crystals, urea crystals or aluminum salts, which raises questions about the specificity of CA074Me. Lysosomal rupture is associated with the release of numerous other enzymes, therefore CA074Me might inhibit other released proteases that acts as the essential signal to activate the NLRP3 inflammasome (Montaser et al., 2002). In fact, cathepsin L-deficient macrophages were also used to show that cholesterol crystals led to a diminished release of IL-1 in comparison with wild-type cells (Duewell et al., 2010). However, the dependence of cholesterol crystal-

Involvement of Microglial Cathepsin B in Pro-Interleukin-1 Processing and Persistent Pain 269

phagolysosomes formed by a fusion of SR-A-mediated phagosomes and primary lysosomes. Therefore, pro-caspase-1 and pro-IL-1 in the cytoplasm may be trapped in these cathepsin B-containing phagolysosomes during their formation triggered by a binding of CGA to SR-A. Furthermore, cathepsin B activates pro-caspase-1 and caspase-1 subsequently proteolytically cleaves pro-IL-1 and pro-IL-18 to their mature forms. Finally, mature IL-1

Cathepsin B deficiency or treatment with a specific inhibitor of cathepsin B, CA074Me, was found to abrogate CGA-induced activation of pro-caspase-1 and subsequent processing of the inactive forms of IL-1 and IL-18 to their mature forms in microglia. Furthermore, the existence of multiple pathways that can induce the proteolytic cleavage of pro-IL-1 and pro-IL-18 in microglia was further demonstrated, probably indicating that there is a backup system for generating these cytokines. CGA activates cathepsin B-dependent but NLRP3 independent pathways for the processing of both IL-1 and IL-18, whereas ATP activates NLRP3-dependent but cathepsin B-independent pathways for their processing. These *in vitro* observations using cultured microglia prompted us to further investigate the role of cathepsin B in chronic pain generation, because both IL-1 and IL-18 are involved in the initiation of inflammatory and pain hypersensitivity (Samad et al., 2001; Sweitzer et al., 2001; Kawasaki et al., 2008; Miyoshi et al., 2008). Furthermore, Inoue's group (Kyushu University) introduced the concept that signals derived from spinal microglia after spinal nerve injury are the core mechanisms underlying neuropathic pain (Tsuda et al., 2003; Coull et al., 2005; Scholz & Woolf, 2007). Following intra-plantar injection of complete Freund's adjuvant (CFA), both hyperalgesia (an augmented pain response to noxious stimulation) and allodynia (a pain produced by normally non-painful stimulation) develop in the injected paw. As expected, cathepsin B deficiency or the intrathecal administration of a specific cathepsin B inhibitor significantly inhibited both CFA-induced mechanical allodynia and thermal hyperalgesia without affecting peripheral inflammation. In contrast, cathepsin Bdeficiency had no significant effect on spinal nerve injury-induced mechanical allodynia. At the same time, mature IL-1 and IL-18 were expressed in the spinal microglia of cathepsin Bdeficient mice following spinal nerve-transection, but not after CFA treatment. Treatment with minocycline, a microglial activation inhibitor, completely prevented the development of CFA-induced mechanical allodynia and thermal hyperalgesia, thus suggesting that microglial activation and inflammatory immune responses in the spinal cord are involved in CFA-induced mechanical allodynia and thermal hyperalgesia (Shan et al., 2007). Therefore, it is considered that peripheral CFA-treatment triggers cathepsin B-dependent caspase-1 activation pathways for the processing of both IL-1 and IL-18 in spinal microglia, leading to pain hypersensitivity (Figure 1). On the other hand, spinal nerve-injury activates cathepsin B-independent pathways for the processing of both IL-1 and IL-18 in spinal microglia. A MMP-9-dependent mechanism has been proposed as an alternative pathway for the proteolytic cleavage of pro-IL-1 in the spinal cord following nerve injury (Kawasaki

Following the peripheral inflammation, IL-1 and IL-18 are secreted from activated spinal microglia in a cathepsin B-dependent manner. IL-1 and IL-18 subsequently induce COX-2 in spinal neurons, leading to the production of prostaglandin E2 (PGE2). PGE2 activates protein kinase A (PKA) through increase in cAMP levels by binding to PGE2 receptors of the

and IL-18 are secreted extracellulary by exocytosis (Figure 1).

**5. Cathepsin B and Inflammatory pain**

et al., 2008).

induced IL-1 release on cathepsin B or L was less pronounced at higher concentrations, thus suggesting the functional redundancy of cathepsin B/L, or the potential presence of additional proteases. Furthermore, Masters et al. (2010) showed that the secretion of IL-1 from bone marrow-derived dendritic cells after phagocytosis of islet amyloid polypeptide, a unique polypeptide constituent of amyloid found in pancreatic islet, was significantly inhibited by a specific inhibitor of either cathepsin B or ROS. Proving the importance of the lysosomal rupture model will require identification of the putative cathepsin B substrate(s) that activate the NLRP3 inflammasome. Detailed analyses are also needed to clarify the involvement of cathepsins other than cathepsin B for activation of the NLRP3 inflammasome. On the other hand, proving the importance of the ROS model will require that the source of the ROS that activate the NLRP3 inflammasome be clarified. Phagocytosed particulates that are too large to be efficiently cleared are likely to induce the production of ROS on their way to lysosomes. Therefore, the lysosomal rupture model could be viewed as forming part of a more general ROS pathway. It is likely that the activation of the NLRP3 is more complex and may require a combination of factors, including both enzymatic activities of cathepsins and ROS activity.

#### **4.2 Alternative mechanisms underlying the cathepsin B-dependent activation of procaspase-1**

There is still evidence suggesting that cathepsin B is directly associated with the proteolytic cleavage of pro-IL-1 in the endosomal/lysosomal system. Cathepsin B can efficiently cleave pro-caspase-11 in a cell-free system even at a neutral pH, but it cleaves pro-caspase-1 only at an acidic pH (Vancompernolle et al., 1998). Furthermore, Hentze et al. (2003) found that cathepsin B is directly involved in the proteolytic cleavage of pro-casapase-1 in THP-1 monocytic cells after stimulation with the microbial toxin nigericin. However, the size of the cleaved fragments of pro-caspase-1 generated by cathepsin B (37 and 40 kDa) is different from the active fragments that are produced by caspase-1 self-processing (10 and 20 kDa). It is possible that the fragments of pro-caspase-1 resulting from the cleavage by cathepsin B may be further cleaved to the active fragments by self-processing. It should also be considered whether cathepsin B is indirectly involved in the activation of pro-caspase-1 though its direct activation of pro-caspase-11, because caspase-11 is known to be play a crucial role in the activation of pro-caspase-1 (Kang et al., 2000).

During the course of experiments to examine the role of cathepsin B in microglial apoptosis, we found that cathepsin B-deficiency abrogated the secretion of IL-1 from microglia following treatment with CGA (Terada et al., 2010). Detailed analyses revealed that cathepsin B-deficiency and CA074Me significantly inhibited the proteolytic processing of pro-IL-1 and secretion of mature IL-1 from microglia following treatment with CGA without affecting the increased production of pro-IL-1. Furthermore, there was no sign of any leakage of cathepsin B in microglia following treatment with CGA. The typical size of the primary lysosomes is below 1 m in diameter, whereas the mean diameter of cathepsin B-containing enlarged lysosomes in CGA-stimulated microglia was 4.2 m. Furthermore, cathepsin B-positive enlarged lysosomes were found to be acidic compartments. These findings are consistent with previous observations that IL-1 and cathepsin D are colocalized within endolysosome-related vesicles, and that the secretion of IL-1 involves the exocytosis of these vesicles in LPS-activated human monocytes (Andrei et al., 1999).

Interestingly, CGA is known to activate microglia through scavenger receptor class-A (SR-A; Hooper et al., 2009). Cathepsin B-containing enlarged lysosomes are considered to be phagolysosomes formed by a fusion of SR-A-mediated phagosomes and primary lysosomes. Therefore, pro-caspase-1 and pro-IL-1 in the cytoplasm may be trapped in these cathepsin B-containing phagolysosomes during their formation triggered by a binding of CGA to SR-A. Furthermore, cathepsin B activates pro-caspase-1 and caspase-1 subsequently proteolytically cleaves pro-IL-1 and pro-IL-18 to their mature forms. Finally, mature IL-1 and IL-18 are secreted extracellulary by exocytosis (Figure 1).

#### **5. Cathepsin B and Inflammatory pain**

Inflammation, Chronic Diseases and Cancer –

268 Cell and Molecular Biology, Immunology and Clinical Bases

induced IL-1 release on cathepsin B or L was less pronounced at higher concentrations, thus suggesting the functional redundancy of cathepsin B/L, or the potential presence of additional proteases. Furthermore, Masters et al. (2010) showed that the secretion of IL-1 from bone marrow-derived dendritic cells after phagocytosis of islet amyloid polypeptide, a unique polypeptide constituent of amyloid found in pancreatic islet, was significantly inhibited by a specific inhibitor of either cathepsin B or ROS. Proving the importance of the lysosomal rupture model will require identification of the putative cathepsin B substrate(s) that activate the NLRP3 inflammasome. Detailed analyses are also needed to clarify the involvement of cathepsins other than cathepsin B for activation of the NLRP3 inflammasome. On the other hand, proving the importance of the ROS model will require that the source of the ROS that activate the NLRP3 inflammasome be clarified. Phagocytosed particulates that are too large to be efficiently cleared are likely to induce the production of ROS on their way to lysosomes. Therefore, the lysosomal rupture model could be viewed as forming part of a more general ROS pathway. It is likely that the activation of the NLRP3 is more complex and may require a combination of factors, including both

**4.2 Alternative mechanisms underlying the cathepsin B-dependent activation of pro-**

There is still evidence suggesting that cathepsin B is directly associated with the proteolytic cleavage of pro-IL-1 in the endosomal/lysosomal system. Cathepsin B can efficiently cleave pro-caspase-11 in a cell-free system even at a neutral pH, but it cleaves pro-caspase-1 only at an acidic pH (Vancompernolle et al., 1998). Furthermore, Hentze et al. (2003) found that cathepsin B is directly involved in the proteolytic cleavage of pro-casapase-1 in THP-1 monocytic cells after stimulation with the microbial toxin nigericin. However, the size of the cleaved fragments of pro-caspase-1 generated by cathepsin B (37 and 40 kDa) is different from the active fragments that are produced by caspase-1 self-processing (10 and 20 kDa). It is possible that the fragments of pro-caspase-1 resulting from the cleavage by cathepsin B may be further cleaved to the active fragments by self-processing. It should also be considered whether cathepsin B is indirectly involved in the activation of pro-caspase-1 though its direct activation of pro-caspase-11, because caspase-11 is known to be play a

During the course of experiments to examine the role of cathepsin B in microglial apoptosis, we found that cathepsin B-deficiency abrogated the secretion of IL-1 from microglia following treatment with CGA (Terada et al., 2010). Detailed analyses revealed that cathepsin B-deficiency and CA074Me significantly inhibited the proteolytic processing of pro-IL-1 and secretion of mature IL-1 from microglia following treatment with CGA without affecting the increased production of pro-IL-1. Furthermore, there was no sign of any leakage of cathepsin B in microglia following treatment with CGA. The typical size of the primary lysosomes is below 1 m in diameter, whereas the mean diameter of cathepsin B-containing enlarged lysosomes in CGA-stimulated microglia was 4.2 m. Furthermore, cathepsin B-positive enlarged lysosomes were found to be acidic compartments. These findings are consistent with previous observations that IL-1 and cathepsin D are colocalized within endolysosome-related vesicles, and that the secretion of IL-1 involves the exocytosis of these vesicles in LPS-activated human monocytes (Andrei et al., 1999). Interestingly, CGA is known to activate microglia through scavenger receptor class-A (SR-A; Hooper et al., 2009). Cathepsin B-containing enlarged lysosomes are considered to be

enzymatic activities of cathepsins and ROS activity.

crucial role in the activation of pro-caspase-1 (Kang et al., 2000).

**caspase-1**

Cathepsin B deficiency or treatment with a specific inhibitor of cathepsin B, CA074Me, was found to abrogate CGA-induced activation of pro-caspase-1 and subsequent processing of the inactive forms of IL-1 and IL-18 to their mature forms in microglia. Furthermore, the existence of multiple pathways that can induce the proteolytic cleavage of pro-IL-1 and pro-IL-18 in microglia was further demonstrated, probably indicating that there is a backup system for generating these cytokines. CGA activates cathepsin B-dependent but NLRP3 independent pathways for the processing of both IL-1 and IL-18, whereas ATP activates NLRP3-dependent but cathepsin B-independent pathways for their processing. These *in vitro* observations using cultured microglia prompted us to further investigate the role of cathepsin B in chronic pain generation, because both IL-1 and IL-18 are involved in the initiation of inflammatory and pain hypersensitivity (Samad et al., 2001; Sweitzer et al., 2001; Kawasaki et al., 2008; Miyoshi et al., 2008). Furthermore, Inoue's group (Kyushu University) introduced the concept that signals derived from spinal microglia after spinal nerve injury are the core mechanisms underlying neuropathic pain (Tsuda et al., 2003; Coull et al., 2005; Scholz & Woolf, 2007). Following intra-plantar injection of complete Freund's adjuvant (CFA), both hyperalgesia (an augmented pain response to noxious stimulation) and allodynia (a pain produced by normally non-painful stimulation) develop in the injected paw. As expected, cathepsin B deficiency or the intrathecal administration of a specific cathepsin B inhibitor significantly inhibited both CFA-induced mechanical allodynia and thermal hyperalgesia without affecting peripheral inflammation. In contrast, cathepsin Bdeficiency had no significant effect on spinal nerve injury-induced mechanical allodynia. At the same time, mature IL-1 and IL-18 were expressed in the spinal microglia of cathepsin Bdeficient mice following spinal nerve-transection, but not after CFA treatment. Treatment with minocycline, a microglial activation inhibitor, completely prevented the development of CFA-induced mechanical allodynia and thermal hyperalgesia, thus suggesting that microglial activation and inflammatory immune responses in the spinal cord are involved in CFA-induced mechanical allodynia and thermal hyperalgesia (Shan et al., 2007). Therefore, it is considered that peripheral CFA-treatment triggers cathepsin B-dependent caspase-1 activation pathways for the processing of both IL-1 and IL-18 in spinal microglia, leading to pain hypersensitivity (Figure 1). On the other hand, spinal nerve-injury activates cathepsin B-independent pathways for the processing of both IL-1 and IL-18 in spinal microglia. A MMP-9-dependent mechanism has been proposed as an alternative pathway for the proteolytic cleavage of pro-IL-1 in the spinal cord following nerve injury (Kawasaki et al., 2008).

Following the peripheral inflammation, IL-1 and IL-18 are secreted from activated spinal microglia in a cathepsin B-dependent manner. IL-1 and IL-18 subsequently induce COX-2 in spinal neurons, leading to the production of prostaglandin E2 (PGE2). PGE2 activates protein kinase A (PKA) through increase in cAMP levels by binding to PGE2 receptors of the

Involvement of Microglial Cathepsin B in Pro-Interleukin-1 Processing and Persistent Pain 271

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G.S., Franchi, L., Nuňez, G., Schnurr, M., Espevik, T., Lien, E., Fitzgerald, K.A., Rock, K.L., Moore, K.J., Wright, S.D., Hornung, V. & Latz, E. (2010). NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol

Lohr, S., Patthy, L., Gonzalez-Zulueta, M., Nikolich, K & Urfer, R. (2004) Identification of cathepsin B as a mediator of neuronal death induced by A activated microglial cells using a functional genomic approach, *Journal of Biological*

involved in the trafficking of TNF--containing vesicles to the plasma membrane in

K.A., Latz, E., Moore, K.J. & Golenbock, D.T. (2008) The NALP3 inflammasome is involved in the innate immune response to amyloid-, *Nature Immunology* Vol. 9

K.A. & Latz, E. (2008). Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilizatiuon, *Nature Immunology* Vol. 9

Mediating caspase-1-dependent interleukin-18 maturation and caspase-1 independent necrosis triggered by the microbial toxin nigericin, *Cell Death and*

chromogranin A-induced microglial stress and neurotoxic cascades, *FEBS Letters*

Srinivasan, A., Tomaselli, K.J., Tornberry, N.A., Moskowitz, M. & Yuang, J. (2000). Dual role of caspase-11 in mediating activation of caspase-1 and caspase-3 under

Corfas, G., Lo, E.H. & Ji, R.-R. (2008). Distinct roles of matrix metalloproteases in

**8. References**

8): e6510.

(No. 8): 857-865.

(No. 8): 847-856.

EP2 subtype. PKA then causes phosphorylation and inhibition of glycine receptors containing the 3 subunits (GlyR3). This disinhibition induces hypersensitivity of pain. IL-1 and IL-18 are also known to directly enhance responses mediated by NMDA receptors.

Fig. 1. Schematic representation of the role of cathepsin B in the initiation of inflammatory pain.

#### **6. Conclusion**

The lysosomal rupture and subsequent leakage of cathepsin B has been proposed as the common molecular basis underlying apoptosis and inflammation. Although a lysosomal rupture has pathological significance, we have demonstrated that cathepsin B is involved in the proteolytic processing of pro-caspase-1 to its active form in the phagolysosomes of microglia, even in the absence of leakage. Therefore, it is considered that cathepsin Bdependency and the mechanism of action depend on the activator or stimulus. The growing understanding of the proteolytic systems of cathepsins in the central nervous system could contribute to the development of protease inhibitors as therapeutic interventions against chronic inflammation-related diseases including chronic pain.

#### **7. Acknowledgment**

This study was supported by Japan and the Technology Agency, CREST, and Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

#### **8. References**

Inflammation, Chronic Diseases and Cancer –

270 Cell and Molecular Biology, Immunology and Clinical Bases

EP2 subtype. PKA then causes phosphorylation and inhibition of glycine receptors containing the 3 subunits (GlyR3). This disinhibition induces hypersensitivity of pain. IL-1 and IL-18 are also known to directly enhance responses mediated by NMDA receptors.

Fig. 1. Schematic representation of the role of cathepsin B in the initiation of inflammatory

The lysosomal rupture and subsequent leakage of cathepsin B has been proposed as the common molecular basis underlying apoptosis and inflammation. Although a lysosomal rupture has pathological significance, we have demonstrated that cathepsin B is involved in the proteolytic processing of pro-caspase-1 to its active form in the phagolysosomes of microglia, even in the absence of leakage. Therefore, it is considered that cathepsin Bdependency and the mechanism of action depend on the activator or stimulus. The growing understanding of the proteolytic systems of cathepsins in the central nervous system could contribute to the development of protease inhibitors as therapeutic interventions against

This study was supported by Japan and the Technology Agency, CREST, and Grants-in-Aid

from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

chronic inflammation-related diseases including chronic pain.

pain.

**6. Conclusion**

**7. Acknowledgment**

**12**

C. E. Pang

*Singapore*

*Singapore National Eye Centre*

**Review of Cytomegalovirus Anterior Uveitis**

Cytomegalovirus (CMV), a member of the herpes family which includes herpes simplex virus (HSV) 1 and 2, varicella zoster virus (VZV), and Epstein-Barr virus (EBV), is ubiquitous worldwide. About 85% of persons above the age of 40 in the general community are seropositive.1 CMV infections manifest more usually in immunocompromised individuals especially in patients with acquired immune deficiency syndrome (AIDS); however more recently, CMV infections have been reported in the immunocompetent person, manifesting as corneal endotheliitis as well as anterior uveitis associated with elevated intraocular pressure (IOP; hypertensive anterior uveitis).2-8 This chapter aims to review the ocular features and treatment of CMV anterior uveitis in the

Viruses are increasingly being implicated as a cause of what was previously considered to be idiopathic ocular inflammations in immunocompetent patients. CMV and HSV have been associated with Possner-Schlossman Syndrome (PSS)4,9,10 and rubella and HSV have been associated with Fuchs Heterochromic Iridocyclitis (FHI).11-13 In various studies, the presence of CMV deoxyribonucleic acid (DNA) has been identified in 41.7% of FHI and

The overlap between the different viruses and the variability in clinical presentations do suggest that ocular manifestations of viral diseases in immunocompetent patients may not be specific to a particular virus but rather a response to the viral infection based on the

These viruses share common characteristics. The core contains a linear double-stranded DNA, which is surrounded by a capsid and an envelope. The envelope is a derivative of the core membrane of the infected cells and consists of lipids with inserted viral glycoprotein. There are specific receptors of the glycoproteins of the envelope that recognize complementary receptors on the host cell membrane and bind to them by adsorption. The envelope and the cell membrane fuse while the viral nucleocapsid enters the cell. The viral proteins are then produced in a cascade. The synthesis of DNA and assembly of capsid take place in the nucleus while the production of infected particles in the cytoplasm leads to destruction of the host cell.15-16 Viral DNA has been detected in monocytes, dendritic cells,

**1. Introduction**

immunocompetent person.

**2. Pathophysiology**

52.8% of PSS eyes.14

individual's genetic make-up and immune status.14

megakaryocytes, and myeloid progenitor cells in the bone marrow.

the early- and late-phase development of neuropathic pain. *Nature Medicine* Vol.14 (No. 3 ): 331-336.

### **Review of Cytomegalovirus Anterior Uveitis**

C. E. Pang *Singapore National Eye Centre Singapore*

#### **1. Introduction**

Inflammation, Chronic Diseases and Cancer –

272 Cell and Molecular Biology, Immunology and Clinical Bases

Kingham, P.J. & Pocock J.M. (2001) . Microglial secreted cathepsin B induces neuronal

Masters, S.L., Dunne, A., Subramanian, S.I., Hull, R.L., Tannahill, G.M., Sharp, F.A., Becker,

Miyoshi, K., Obata, K., Kondo, T., Okamura, H. & Noguchi, K. (2008). Interleukin-18 -

Montaser, M., Lalmanach, G. & Mach, L. (2002). CA-074, but not its methyl ester CA-074Me,

Mueller-Steiner, S., Zhou, Y., Arai, H., Roberson, E.D., Sun, B., Chen, J., Wang, X., Yu, G.,

Samad, T.A., Moore, K.A., Sapirstein, A., Billet, S., Allchorne, A., Poole, S., Bonventre, J.V.,

Scholz, J. & Woolf, C.J. (2007). The neuropathic pain triad: neurons, immune cells and glia,

Shan, S., Qi-Liang, M.-Y., Hong, C., Tingting, L., Mei, H., Haili, P., Yan-Qing, W., Zhi-Qi, Z.,

Sweitzer, S., Martin, D. & Deleo, J.A. (2001). Intratechal interleukin-1 receptor antagonist in

Tsuda, M., Shigemoto-Mogami, Y., Koizumi, S., Mizokoshi, A., Kohsaka, S., Salter, M.W. &

Vancompernolle, K., Van Herreweghe, F., Pynaert, G., Van de Craen, M., De Vos, K., Totty,

Wendt, W., Schulten, R., Stichel, C.C. & Lübbert, H. (2009). Intra- versus extracellular

monoarthritis? *Neurobiology of Disease* Vol. 26 (No. 3): 558-568.

apoptosis, *Journal of Neurochemistry* Vol. 76 (No. 5): 1475-1484.

diabetes, *Nature Immunology* Vol. 11 (No. 10): 897-904.

*Nature Neuroscience* Vol. 10 (No. 11): 1361-1368.

stimulated microglia, *Glia* Vol. 58 (No. 1): 114-124.

*Letters* Vol. 438 (No. 3): 150-158.

after nerve injury, *Nature* Vol. 424 (No. 6950): 778-783.

model, *Journal of Neurochemistry* Vol. 110 (No. 6): 1931-1941.

(No. 3 ): 331-336.

12787.

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475.

(No. 7-8): 1305-1308.

the early- and late-phase development of neuropathic pain. *Nature Medicine* Vol.14

C., Franchi, L., Yoshihara, E., Chen, Z., Mullooly, N., Mielke L.A., Harris, J., Coll, R.C., Mills, K.H.G., Mok, K.H., Newsholme, P., Nuňez, G., Yodoi, J., Kahn, S.E., Lavelle, E.D. & O'Neill L.A.J. (2010). Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1 in type 2

mediated microglia/astrocyte interaction in the spinal cord enhances neuropathic pain procesng after nerve injury, Journal of Neuroscience Vol. 28 (No. 48): 12775-

is a selective inhibitor of cathepsin B within living cells, *Biological Chemistry* Vol. 383

Esposito, L., Mucke, L. & Gan, L. (2006). Antiamyloidogenic and neuroprotective functions of cathepsin B: Implications for Alzheimer's disease, *Neuron* Vol. 51 (No.

Woolf, C.J. (2001). Interleukin-1-mediated induction of cox-2 in the CNS contributes to inflammatory pain hypersensitivity, Nature Vol. 410 (No. 6827): 471-

Yu-Qiu, Z. (2007). Is functional state of spinal microglia involved in the antiallodynic and anti-hyperalgesic effects of electroacupuncture in rat model of

Combination with soluble tumor necrosis factor recepyor exhibits an anti- allodynic action in a rat model of neuropathic pain. *Neuroscience* Vol. 103 (No. 2): 529-539. Terada, K., Yamada, J., Hayashi, Y., Wu, Z. & Nakanishi, H. (2010). Involvement of

cathepsin B in the processing and secretion of interlukin-1- in chromogranin A -

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effects of microglia-derived cysteine proteases in a conditioned medium transfer

Cytomegalovirus (CMV), a member of the herpes family which includes herpes simplex virus (HSV) 1 and 2, varicella zoster virus (VZV), and Epstein-Barr virus (EBV), is ubiquitous worldwide. About 85% of persons above the age of 40 in the general community are seropositive.1 CMV infections manifest more usually in immunocompromised individuals especially in patients with acquired immune deficiency syndrome (AIDS); however more recently, CMV infections have been reported in the immunocompetent person, manifesting as corneal endotheliitis as well as anterior uveitis associated with elevated intraocular pressure (IOP; hypertensive anterior uveitis).2-8 This chapter aims to review the ocular features and treatment of CMV anterior uveitis in the immunocompetent person.

#### **2. Pathophysiology**

Viruses are increasingly being implicated as a cause of what was previously considered to be idiopathic ocular inflammations in immunocompetent patients. CMV and HSV have been associated with Possner-Schlossman Syndrome (PSS)4,9,10 and rubella and HSV have been associated with Fuchs Heterochromic Iridocyclitis (FHI).11-13 In various studies, the presence of CMV deoxyribonucleic acid (DNA) has been identified in 41.7% of FHI and 52.8% of PSS eyes.14

The overlap between the different viruses and the variability in clinical presentations do suggest that ocular manifestations of viral diseases in immunocompetent patients may not be specific to a particular virus but rather a response to the viral infection based on the individual's genetic make-up and immune status.14

These viruses share common characteristics. The core contains a linear double-stranded DNA, which is surrounded by a capsid and an envelope. The envelope is a derivative of the core membrane of the infected cells and consists of lipids with inserted viral glycoprotein. There are specific receptors of the glycoproteins of the envelope that recognize complementary receptors on the host cell membrane and bind to them by adsorption. The envelope and the cell membrane fuse while the viral nucleocapsid enters the cell. The viral proteins are then produced in a cascade. The synthesis of DNA and assembly of capsid take place in the nucleus while the production of infected particles in the cytoplasm leads to destruction of the host cell.15-16 Viral DNA has been detected in monocytes, dendritic cells, megakaryocytes, and myeloid progenitor cells in the bone marrow.

Review of Cytomegalovirus Anterior Uveitis 275

and fine to medium keratic precipitates either in a ring or linear pattern. Diffuse and patchy

FHI may be unilateral or bilateral. The main symptom is blurred vision. Clinical features include elevated IOP, anterior chamber cells (grades 1 to 2+), fine feathery, diffuse keratic

From literature data to date, 110 eyes of 106 non-HIV positive patients with CMV anterior segment infection have been described4-9 of which 27 eyes of 24 patients had endotheliitis, 57 eyes had acute recurrent anterior uveitis and the remaining had chronic anterior uveitis. All

The usefulness of aqueous humor sampling has been established in both anterior uveitis and posterior uveitis. A number of recent studies have shown the benefit of aqueous humor polymerase chain reaction (PCR) for the diagnosis of infectious uveitis and revealed a good degree of concordance between intraocular antibody and real-time PCR. Testing for DNA tends to be positive at the outset (and/or early at reactivation) and antibody testing can be positive at any point in time.25 A positive test result indicates at the most that the virus may be involved, but is by no means conclusive. A negative test does not exclude the herpes

A minimum of 100 μl aqueous should be obtained via a diagnostic anterior chamber paracentesis and the samples must be delivered to the laboratory at 18 to 25 degrees Celsius within one hour or stored and transported at 2 to 4 degrees Celsius within 24 hours. The samples should be aliquoted immediately on arrival at the laboratory and used for DNA extraction immediately or kept at −20 degrees Celsius and used for DNA extraction within

Diagnostic anterior chamber paracentesis should involve the use of a 27-gauge needle inserted into the temporal, perilimbal, inferior one-third of the cornea; directed downwards while avoiding the lens. This has been shown to be a simple and safe office procedure.26 Identification of the CMV virus makes it possible to institute the appropriate antiviral

Management of CMV anterior uveitis may involve the use of topical non-steroidal antiinflammatory agents, topical steroids, topical anti-glaucoma medications and topical or systemic antivirals. Patients should also be co-managed by the infectious disease physician to exclude systemic cytomegalovirus infections and other forms of immunocompromised

Topical non-steroidal anti-inflammatory agents have been employed in the immediate setting to reduce ocular inflammation and treat the patient symptomatically. This is especially useful when the use of topical steroids is delayed intentionally till after the diagnostic anterior chamber paracentesis to allow better yield of viral DNA load when

Topical steroids alone may be able to reduce the inflammation and IOP in a minority of cases. More commonly, combination with anti-glaucoma medication will give rise to more

the 83 anterior uveitis eyes and 24 of the endotheliitis eyes had ocular hypertension.

iris stromal atrophy is common.

virus as a cause of the uveitis either.

**4. Diagnosis**

one week.

medication.

states.

**5. Treatment**

performing aqueous sampling.

precipitates and diffuse "moth-eaten" iris atrophy.

Herpetic viruses are well known for their ability to cause keratitis, anterior uveitis, scleritis, and retinitis.17-19 Anterior uveitis is unilateral and typically has a chronic and/or recurrent character. Intraocular pressure (IOP) elevation, keratitis, endotheliitis, and stromal iris atrophy commonly accompany it. The iris stroma is infiltrated with lymphocytes, and an increase in IOP is typically seen on activation of the uveitis, probably due to an outflow obstruction. This outflow obstruction can be explained by both a viral trabeculitis with swelling of the trabecular cells and an obstruction of the trabecular meshwork by inflammatory debris.18-19

The pathogenesis of CMV uveitis, although not completely understood, is believed to include various mechanisms such as viral replication, ischemic vasculitis, lymphocytic infiltration of the iris stroma or intraocular nerves. In addition, persistent and recurrent viral infection may cause an inflammatory reaction manifested as uveitis, or can trigger the immune system itself against viral antigens, eventually causing tissue and organ inflammation and damage.20-24

#### **3. Ocular features**

CMV in the anterior segment is a newly described entity that occurs even in people who are not infected with the human immunodeficiency virus (HIV).

CMV infection can cause a spectrum of ocular manifestations, varying in severity from a mild self-limiting iritis with sector iris atrophy,6 to features of PSS,4-9 to the more chronic form resembling FHI, or even corneal endotheliitis.7 Two common features seen in these eyes with CMV uveitis are elevated IOP and iris atrophy. These features are consistent with the finding of CMV in the smooth muscle cells of the iris, the ciliary body, and the endothelial cells of the Schlemm canal.17

In an analysis of published studies of non-HIV, CMV-associated anterior uveitis in immunocompetent patients, researchers found clinical presentations including endotheliitis, recurrent acute anterior uveitis and ocular hypertension, and chronic anterior uveitis.12 Eyes with such CMV-associated anterior uveitis usually have no corneal scars, no posterior synechiae, no flare or fibrin and no posterior segment involvement.

Corneal endotheliitis may be unilateral or bilateral, typically causes blurred vision and is associated with corneal edema, Descemet's folds, as well as fine and medium keratic precipitates that may be pigmented. Other associated clinical signs include iris atrophy, mild anterior chamber activity, reduced endothelial cell count, elevated IOP, coin-like lesions and the "owl eye sign" on confocal microscopy, representing inclusion bodies and macrophages.

As mentioned, CMV can present clinically as PSS or FHI. PSS and FHI are 2 separate clinical entities that are similar in some aspects, but yet have important distinguishing features. Both have elevated intraocular pressures during the episodes of acute iritis (hypertensive anterior uveitis). Both have only a mild non-granulomatous anterior chamber activity with no posterior synechiae formation. However, PSS is an acute, intermittent, recurrent glaucomatocyclitic crisis with few keratic precipitates, while FHI is a chronic iridocyclitis with diffuse, fine, stellate keratic precipitates and may have vitritis. Exact aetiology of both conditions is still arguable although the consensus is that they are attributable to viruses including CMV as mentioned above.

PSS is usually unilateral, with symptoms of redness, blurring, haloes and unilateral headache. Clinical features include elevated IOP, anterior chamber cells (grades ½ to 2+) and fine to medium keratic precipitates either in a ring or linear pattern. Diffuse and patchy iris stromal atrophy is common.

FHI may be unilateral or bilateral. The main symptom is blurred vision. Clinical features include elevated IOP, anterior chamber cells (grades 1 to 2+), fine feathery, diffuse keratic precipitates and diffuse "moth-eaten" iris atrophy.

From literature data to date, 110 eyes of 106 non-HIV positive patients with CMV anterior segment infection have been described4-9 of which 27 eyes of 24 patients had endotheliitis, 57 eyes had acute recurrent anterior uveitis and the remaining had chronic anterior uveitis. All the 83 anterior uveitis eyes and 24 of the endotheliitis eyes had ocular hypertension.

#### **4. Diagnosis**

Inflammation, Chronic Diseases and Cancer –

274 Cell and Molecular Biology, Immunology and Clinical Bases

Herpetic viruses are well known for their ability to cause keratitis, anterior uveitis, scleritis, and retinitis.17-19 Anterior uveitis is unilateral and typically has a chronic and/or recurrent character. Intraocular pressure (IOP) elevation, keratitis, endotheliitis, and stromal iris atrophy commonly accompany it. The iris stroma is infiltrated with lymphocytes, and an increase in IOP is typically seen on activation of the uveitis, probably due to an outflow obstruction. This outflow obstruction can be explained by both a viral trabeculitis with swelling of the trabecular cells and an obstruction of the trabecular meshwork by

The pathogenesis of CMV uveitis, although not completely understood, is believed to include various mechanisms such as viral replication, ischemic vasculitis, lymphocytic infiltration of the iris stroma or intraocular nerves. In addition, persistent and recurrent viral infection may cause an inflammatory reaction manifested as uveitis, or can trigger the immune system itself against viral antigens, eventually causing tissue and organ

CMV in the anterior segment is a newly described entity that occurs even in people who are

CMV infection can cause a spectrum of ocular manifestations, varying in severity from a mild self-limiting iritis with sector iris atrophy,6 to features of PSS,4-9 to the more chronic form resembling FHI, or even corneal endotheliitis.7 Two common features seen in these eyes with CMV uveitis are elevated IOP and iris atrophy. These features are consistent with the finding of CMV in the smooth muscle cells of the iris, the ciliary body, and the

In an analysis of published studies of non-HIV, CMV-associated anterior uveitis in immunocompetent patients, researchers found clinical presentations including endotheliitis, recurrent acute anterior uveitis and ocular hypertension, and chronic anterior uveitis.12 Eyes with such CMV-associated anterior uveitis usually have no corneal scars, no posterior

Corneal endotheliitis may be unilateral or bilateral, typically causes blurred vision and is associated with corneal edema, Descemet's folds, as well as fine and medium keratic precipitates that may be pigmented. Other associated clinical signs include iris atrophy, mild anterior chamber activity, reduced endothelial cell count, elevated IOP, coin-like lesions and the "owl eye sign" on confocal microscopy, representing inclusion bodies and

As mentioned, CMV can present clinically as PSS or FHI. PSS and FHI are 2 separate clinical entities that are similar in some aspects, but yet have important distinguishing features. Both have elevated intraocular pressures during the episodes of acute iritis (hypertensive anterior uveitis). Both have only a mild non-granulomatous anterior chamber activity with no posterior synechiae formation. However, PSS is an acute, intermittent, recurrent glaucomatocyclitic crisis with few keratic precipitates, while FHI is a chronic iridocyclitis with diffuse, fine, stellate keratic precipitates and may have vitritis. Exact aetiology of both conditions is still arguable although the consensus is that they are attributable to viruses

PSS is usually unilateral, with symptoms of redness, blurring, haloes and unilateral headache. Clinical features include elevated IOP, anterior chamber cells (grades ½ to 2+)

not infected with the human immunodeficiency virus (HIV).

synechiae, no flare or fibrin and no posterior segment involvement.

inflammatory debris.18-19

inflammation and damage.20-24

endothelial cells of the Schlemm canal.17

including CMV as mentioned above.

**3. Ocular features**

macrophages.

The usefulness of aqueous humor sampling has been established in both anterior uveitis and posterior uveitis. A number of recent studies have shown the benefit of aqueous humor polymerase chain reaction (PCR) for the diagnosis of infectious uveitis and revealed a good degree of concordance between intraocular antibody and real-time PCR. Testing for DNA tends to be positive at the outset (and/or early at reactivation) and antibody testing can be positive at any point in time.25 A positive test result indicates at the most that the virus may be involved, but is by no means conclusive. A negative test does not exclude the herpes virus as a cause of the uveitis either.

A minimum of 100 μl aqueous should be obtained via a diagnostic anterior chamber paracentesis and the samples must be delivered to the laboratory at 18 to 25 degrees Celsius within one hour or stored and transported at 2 to 4 degrees Celsius within 24 hours. The samples should be aliquoted immediately on arrival at the laboratory and used for DNA extraction immediately or kept at −20 degrees Celsius and used for DNA extraction within one week.

Diagnostic anterior chamber paracentesis should involve the use of a 27-gauge needle inserted into the temporal, perilimbal, inferior one-third of the cornea; directed downwards while avoiding the lens. This has been shown to be a simple and safe office procedure.26 Identification of the CMV virus makes it possible to institute the appropriate antiviral medication.

#### **5. Treatment**

Management of CMV anterior uveitis may involve the use of topical non-steroidal antiinflammatory agents, topical steroids, topical anti-glaucoma medications and topical or systemic antivirals. Patients should also be co-managed by the infectious disease physician to exclude systemic cytomegalovirus infections and other forms of immunocompromised states.

Topical non-steroidal anti-inflammatory agents have been employed in the immediate setting to reduce ocular inflammation and treat the patient symptomatically. This is especially useful when the use of topical steroids is delayed intentionally till after the diagnostic anterior chamber paracentesis to allow better yield of viral DNA load when performing aqueous sampling.

Topical steroids alone may be able to reduce the inflammation and IOP in a minority of cases. More commonly, combination with anti-glaucoma medication will give rise to more

Review of Cytomegalovirus Anterior Uveitis 277

[9] Teoh SB, Thean L, Koay E. Cytomegalovirus in aetiology of Posner-Schlossman

[10] Yamamoto S, Pavan-Langston D, Tada R, et al. Possible role of herpes simplex virus in the origin of Posner-Schlossman syndrome. Am J Ophthalmol 1995; 119: 796-798. [11] Baraquet IS, Li Q, Wang Y, O'Brien TP, et al. Herpes simplex virus DNA identification

[12] Quentin CD, Reiber H. Fuchs heterochromic cyclitis: rubella virus antibodies ans

[13] de Groot-Mijnes JDF, de Visser L, Rothova A, et al. Rubella virus is associated with Fuchs heterochromic iridocyclitis. Am J Ophthalmol 2006; 141: 212-214. [14] Chee SP, Bascal K, Jap A, et al. Clinical features of cytomegalovirus in anterior uveitis in

[15] Roizman B: Herpesviridae. In: Fields BN, Knipe DM, Howley PM, eds: Virology, 3rd ed.

[16] Spring SB, Roizman B: Herpes Simplex Virus products in productive and abortive

[17] B. Daicker,B. Cytomegalovirus panuveitis with infection of corneo-trabecular

[18] Heiligenhaus A, Helbig H, Fiedler M. Herpesviruses. In: C.S. Foster and A.T. Vitale,

[19] Holland EJ, Mozayeni RM, Schwartz GS. Herpes simplex keratitis. In: J.H. Krachmer,

[21] Sundmacher R and Neumann-Haefelin D: Herpes simplex virus isolations from the

[23] Liesegang TJ: Corneal complications from herpes zoster ophthalmicus. Ophthalmology

[25] De Groot-Mijnes J, Rothova A, van Loon AM et al., Polymerase chain reaction and

[26] Van der Lelij A, Rothova A, Diagnostic anterior chamber paracentesis in uveitis; a safe

[27] Sira M, Murray PI. Treatment of cytomegalovirus anterior uveitis with oral valaciclovir.

[24] Kijlstra A: The value of laboratory testing in uveitis. Eye 1990; 4: 732-736.

infectious uveitis, Am J Ophthalmol 2006; 141: 313–318.

procedure?, Br J Ophthalmol 1997; 81: 976–979.

Ocul Immunol Inflamm 2007; 15: 31–2.

Clinical Diagnosis and Management, Mosby, St. Louis 1997: 1191–1223. [20] Liesegang TJ: Ocular herpes simplex infection: pathogenesis and current therapy. Mayo

infection. III. Differentiation of infectious virus derived from nucleus and

Editors, Diagnosis and Treatment of Uveitis, W.B. Saunders, Philadelphia 2002:

M.J. Mannis and E.J. Holland, Editors, Cornea. Vol. 2. Cornea and External Disease:

aqueous of patients suffering from local iritis, endotheliitis, and prolonged disciform keratitis with glaucoma. Klin Monatsbl Augenheilkd 1979; 175: 488-501. [22] Pavan-Langston D: Herpes Zoster Ophthalmicus: Medical and surgical management. In

Spaeth G, Katz L, Parker KW (eds): Current therapy in ophthalmic surgery.

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genome in aqueous humor. Am J Ophthalmol 2004; 138: 46-54.

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cytoplasm with respect to stability and size. J Virol 1968; 2: 979.

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effective lowering of IOP. However, steroids may cause steroid-induced glaucoma as a side effect. This is especially seen in patients who are on steroids for a long duration.

Glaucoma therapy can be initiated in a stepwise manner in the following order, unless there are any contraindications to the medication: β-blocker, α-2 agonists, topical acetazolamide, and lastly, prostaglandin analogs. If the IOP exceeds 40 mm Hg, systemic acetazolamide can be considered. The medications when tailed down should be reduced in a stepwise manner as well, but in reverse order.

Mydriatics or cycloplegics can be used as an adjunct to relieve any ciliary spasm, stabilize the blood aqueous barrier and assist in fundal evaluation.

The main mode of antiviral therapy in these eyes has been systemic ganciclovir or valganciclovir.5-8,14,27-28 Some authors have also used intravitreal injections of ganciclovir28 or ganciclovir gel14 with variable outcomes. In many cases, the inflammation resolved with therapy, only to recur on cessation of treatment.5-7 Although there is no consensus as to which route of administration is most suitable, topical ganciclovir gel has been shown to produce less number of recurrences as compared to systemic gangciclovir.29

Response to therapy in eyes with chronic anterior uveitis can be defined as the clinical resolution of anterior chamber cells and keratic precipitates and good IOP control with or without glaucoma medications while on treatment. In eyes with acute recurrent anterior uveitis, response can be defined as absence of recurrence of inflammation and normalisation of IOP without glaucoma medications other than that given during the acute episode while on treatment.

All treated eyes responded to therapy, in terms of the severity of the inflammation and IOP control, to the initial antiviral treatment. However, 77.7% relapsed within eight months after stopping treatment, according to the experience of some authors.6,29 This indicates that CMV anterior uveitis may require a longer period of treatment, perhaps similar to that of herpetic uveitis or even other methods of treatment.

#### **6. References**

276 Cell and Molecular Biology, Immunology and Clinical Bases

effective lowering of IOP. However, steroids may cause steroid-induced glaucoma as a side

Glaucoma therapy can be initiated in a stepwise manner in the following order, unless there are any contraindications to the medication: β-blocker, α-2 agonists, topical acetazolamide, and lastly, prostaglandin analogs. If the IOP exceeds 40 mm Hg, systemic acetazolamide can be considered. The medications when tailed down should be reduced in a stepwise manner

Mydriatics or cycloplegics can be used as an adjunct to relieve any ciliary spasm, stabilize

The main mode of antiviral therapy in these eyes has been systemic ganciclovir or valganciclovir.5-8,14,27-28 Some authors have also used intravitreal injections of ganciclovir28 or ganciclovir gel14 with variable outcomes. In many cases, the inflammation resolved with therapy, only to recur on cessation of treatment.5-7 Although there is no consensus as to which route of administration is most suitable, topical ganciclovir gel has been shown to

Response to therapy in eyes with chronic anterior uveitis can be defined as the clinical resolution of anterior chamber cells and keratic precipitates and good IOP control with or without glaucoma medications while on treatment. In eyes with acute recurrent anterior uveitis, response can be defined as absence of recurrence of inflammation and normalisation of IOP without glaucoma medications other than that given during the acute episode while

All treated eyes responded to therapy, in terms of the severity of the inflammation and IOP control, to the initial antiviral treatment. However, 77.7% relapsed within eight months after stopping treatment, according to the experience of some authors.6,29 This indicates that CMV anterior uveitis may require a longer period of treatment, perhaps similar to that of herpetic

[1] Yoser SL, Forster DJ and Rao NA. Systemic viral infections and their retinal and

[2] Koizumi N, Suzuki T, Uno T, et al. Cytomegalovirus as an etiologic factor in corneal

[3] Teoh SB, Thean L and Koay E. Cytomegalovirus in aetiology of Posner-Schlossman

[4] Bloch-Michel E, Dussaix E, Cerqueti P, et al. Possible role of cytomegalovirus infection in

[5] De Schryver I, Rozenberg F, Cassoux N, et al., Diagnosis and treatment of

[6] Markomichelakis NN, Canakis C, Zafirakis P, et al. Cytomegalovirus as a cause of anterior uveitis with sectoral iris atrophy, Ophthalmology 2002; 109: 879–882. [7] Van Boxtel LA, van der Lelij A, van der Meer J. Cytomegalovirus as a cause of anterior uveitis in immunocompetent patients, Ophthalmology 2007: 114: 1358–1362. [8] Chee SP, Bascal K, Jap A, et al. Corneal endotheliitis associated with evidence of

cytomegalovirus infection, Ophthalmology 2007; 114: 798–803.

syndrome: evidence from quantitative polymerase chain reaction, Eye 2005; 19:

the etiology of the Posner-Schlossmann syndrome, Int Ophthalmol 1987; 11: 95–96.

cytomegalovirus iridocyclitis without retinal necrosis, Br J Ophthalmol 2006; 90:

choroidal manifestations, Surv Ophthalmol 1993; 37: 313–352.

endotheliitis. Ophthalmology. 2008 ; 115(2): 292-297.

effect. This is especially seen in patients who are on steroids for a long duration.

produce less number of recurrences as compared to systemic gangciclovir.29

the blood aqueous barrier and assist in fundal evaluation.

uveitis or even other methods of treatment.

as well, but in reverse order.

on treatment.

**6. References**

1338–1340.

852–855.

**13**

*Sweden*

**"Suppressor of Cytokine Signalling"**

Berit Carow and Martin E. Rottenberg

**Molecules in Infection and Inflammation**

*Dept. of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm*

Cytokines are messengers that coordinate the development and function of leukocytes and therefore are indispensable for the initiation, maintenance and termination of all types of immune responses. A tight control of cytokine functions is crucial for both, the control of infections and the prevention of infection-associated immunopathology. Different intracellular mechanisms of cytokine signal inhibition are involved in the regulation of innate and adaptive immune responses. Among these, the family of suppressor of cytokine signalling (SOCS) proteins identified more than a decade ago (Endo *et al.* 1997; Naka *et al.* 1997; Starr *et al.* 1997) are non-redundant negative feedback inhibitors of both proinflammatory but also of anti-inflammatory cytokine responses (Kubo *et al.* 2003; Yoshimura

Type I and type II cytokine receptors do not possess a cytoplasmic kinase activity and therefore are dependent on associated Janus kinases (JAKs). JAKs conduct the signal of many cytokines including many interleukins (IL), all interferons (IFN) and hemopoietins. Activated JAKs cross-phosphorylate themselves and phosphorylate the associated cytokine receptor creating binding sites for proteins that contain phosphotyrosine binding SH2 domains. The SH2 domain of signal transducers and activators of transcription (STATs) then binds to the phosphorylated receptors. Recruited STATs get phosphorylated by the adjacent JAKs, and act as binding sites for the SH2 domain of another STATs, which also will be phosphorylated. The STAT dimer translocates into the nucleus where it acts as a transcription factor activating the transcription of specific genes. The diversity of STATmediated intracellular pathways is due to the presence of 7 STATs, activated by different receptors. Moreover, activated STAT dimers act as homo- or heterodimers, which increases

the diversity of target promoters and thereby of gene patterns that can be activated.

The JAK-STAT pathway can be negatively regulated at different stages: protein tyrosine phosphatases remove phosphates from cytokine receptors and activated STATs, whereas PIAS (protein inhibitors of activated STATs) act in the nucleus (Leonard & O'Shea 1998; Krebs & Hilton 2001; Hebenstreit *et al.* 2005; Shuai 2006). In this chapter we will focus on the role of another inhibitor of the JAK-STAT pathway, the SOCS proteins. The importance of these proteins is evidenced by the fact that mice deficient for some of the SOCS proteins demonstrated a non-redundant role of SOCS proteins in regulating the immune system

**1. Introduction**

*et al.* 2007).

(Alexander 2002).

### **"Suppressor of Cytokine Signalling" Molecules in Infection and Inflammation**

Berit Carow and Martin E. Rottenberg

*Dept. of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm Sweden*

#### **1. Introduction**

Inflammation, Chronic Diseases and Cancer –

278 Cell and Molecular Biology, Immunology and Clinical Bases

[28] Chung RS, Chua CN. Intravitreal ganciclovir injections in aqueous cytomegalovirus

[29] Chee SP, Jap A. Cytomegalovirus anterior uveitis: outcome of treatment. Br J

DNA positive hypertensive iritis. Eye 2006; 20: 1080.

Ophthalmol. 2010; 94:1648-52.

Cytokines are messengers that coordinate the development and function of leukocytes and therefore are indispensable for the initiation, maintenance and termination of all types of immune responses. A tight control of cytokine functions is crucial for both, the control of infections and the prevention of infection-associated immunopathology. Different intracellular mechanisms of cytokine signal inhibition are involved in the regulation of innate and adaptive immune responses. Among these, the family of suppressor of cytokine signalling (SOCS) proteins identified more than a decade ago (Endo *et al.* 1997; Naka *et al.* 1997; Starr *et al.* 1997) are non-redundant negative feedback inhibitors of both proinflammatory but also of anti-inflammatory cytokine responses (Kubo *et al.* 2003; Yoshimura *et al.* 2007).

Type I and type II cytokine receptors do not possess a cytoplasmic kinase activity and therefore are dependent on associated Janus kinases (JAKs). JAKs conduct the signal of many cytokines including many interleukins (IL), all interferons (IFN) and hemopoietins. Activated JAKs cross-phosphorylate themselves and phosphorylate the associated cytokine receptor creating binding sites for proteins that contain phosphotyrosine binding SH2 domains. The SH2 domain of signal transducers and activators of transcription (STATs) then binds to the phosphorylated receptors. Recruited STATs get phosphorylated by the adjacent JAKs, and act as binding sites for the SH2 domain of another STATs, which also will be phosphorylated. The STAT dimer translocates into the nucleus where it acts as a transcription factor activating the transcription of specific genes. The diversity of STATmediated intracellular pathways is due to the presence of 7 STATs, activated by different receptors. Moreover, activated STAT dimers act as homo- or heterodimers, which increases the diversity of target promoters and thereby of gene patterns that can be activated.

The JAK-STAT pathway can be negatively regulated at different stages: protein tyrosine phosphatases remove phosphates from cytokine receptors and activated STATs, whereas PIAS (protein inhibitors of activated STATs) act in the nucleus (Leonard & O'Shea 1998; Krebs & Hilton 2001; Hebenstreit *et al.* 2005; Shuai 2006). In this chapter we will focus on the role of another inhibitor of the JAK-STAT pathway, the SOCS proteins. The importance of these proteins is evidenced by the fact that mice deficient for some of the SOCS proteins demonstrated a non-redundant role of SOCS proteins in regulating the immune system (Alexander 2002).

"Suppressor of Cytokine Signalling" Molecules in Infection and Inflammation 281

Nicholson *et al.* 2000; Sasaki *et al.* 2000; Hortner *et al.* 2002a; Hortner *et al.* 2002b) and cytokine receptors IL2R and IL-12R1(Cohney *et al.* 1999; Yamamoto *et al.* 2003). Possible interactions of SOCS1 and SOCS3 with Toll-like Receptor (TLR) signalling have been

SOCS1 is expressed constitutively in the thymus, spleen, lung and testes of mice. SOCS1 mRNA expression can be rapidly induced by many cytokines, especially IFNs, and serves as a classical feed back loop inhibiting its inducing pathway (Naka *et al.* 1997; Starr *et al.* 1997). Importantly, SOCS1 mRNA expression increases even in response to microbial molecules such as LPS, Pam3Cys and CpG oligonucleotides that signal via TLR (Dalpke *et al.* 2001; Alexander 2002; Fujimoto & Naka 2003; Dennis *et al.* 2006). Furthermore, hormones like insulin (Emanuelli *et al.* 2000), cardiotrophin (Hamanaka *et al.* 2001) or glucocorticoids

SOCS1-/- animals die within 3 weeks after birth due to fatty degeneration and necrosis of the liver (Naka *et al.* 1998; Starr *et al.* 1998).  At least in part, the spontaneous inflammatory disease is thought to be due to IFN- hyper-responsiveness of SOCS1-deficient tissue, because SOCS1-/-IFN--/- as well as SOCS1-/-STAT1-/- mice survive healthy until adulthood (Alexander *et al.* 1999; Marine *et al.* 1999b). The importance of IFN- was further apparent in mice heterozygous for IFN- and lacking SOCS1 that survived until 5 month of age before succumbing with myocardis and polymyositis (Metcalf *et al.* 2000). SOCS1 mice, treated with neutralizing antibodies from birth, died of the same phenotype reaching adulthood (Bullen *et al.* 2001). A similar expected protection from lethality was observed in SOCS1-/ crossed with IFN-R-/- and STAT1-/- mice. Furthermore, RAG2-/-SOCS1-/- are healthy to at least 3 month of age, implicating that IFN- secreted by T and/ or NKT cells might be responsible for the tremendous inflammation observed (Marine *et al.* 1999b). In fact, depletion of NKT cell by antibody-treatment significantly increased the survival of mice. Surprisingly, mice with a conditional knock down for SOCS1 in T and NKT cells did not display any of the SOCS1-/- pathologies, indicating that a deletion of SOCS1 in T and NKT cells is not sufficient for the hyper-inflammation but that this was due an uncontrolled response of myeloid cells together with an excessive neonatal IFN- release (Chong *et al.* 2003). The fact that myeloid cells are involved was demonstrated by the lethality of chimeric mice, which received SOCS1-/- bone marrow after irradiation (Metcalf *et al.* 2003). On the other hand, T cells were also required for SOCS1-mediated lethality since SOCS1-/- mice with a SOCS1 transgene expressed only by T cells survived. Interestingly, the survival of SOCS1-/- mice also increased when back-crossed to IFNar1-/- mice (lacking one of the IFN- R subunits) indicating that even type I IFNs in part contribute to the lethal inflammation of SOCS1 deficient animals. Altogether, SOCS1 lethality is mainly due to both an exacerbated secretion of IFN- by T and NKT cells during the neonatal period and to a hyper-response of myeloid cells to IFN-, but IFN--independent immune responses such as

(Bhattacharyya *et al.* 2011) have been shown to stimulate SOCS1 expression.

type I IFN and others discussed below are also likely to contribute to it.

The use of conditional knock down mice, as well as in vitro experiments with different cell populations allowed a more precise definition of the role of SOCS1 in different cell lineages. A combination of SOCS1 conditional knock downs in hematopoetic cells using the cre-lox technology confirmed that SOCS1 deficiency in both T cells and macrophages

described and will be discussed later.

**3. SOCS1**

SOCS proteins play a role in balancing immune functions at different levels, including the differentiation of immune cell populations and their activation by environmental stimuli. Both deletion and over-expression of SOCS proteins in animal models provided insights into their importance of regulating the responsiveness to cytokines. Accumulated today's knowledge on the immunobiology of SOCS proteins convert them into new potential targets for treatment of inflammatory diseases but might also help to improve infection control.

### **2. SOCS at the molecular level**

The family of SOCS proteins consists of 8 members (cytokine inducible SH2 protein, CIS, SOCS1-7), which share a central modulator organization with a SH2 domain, a C-terminal SOCS box and an amino-terminal domain of variable length (fig. 1). The SOCS-box was shown to interact with elongin A and B, cullin 5 and ring box. This complex acts as an E3 ubiquitin ligase, initiating ubiquitination in other words the covalent binding of ubiquitin to target proteins, which is followed by the proteosomal degradation of bound signalling complexes as JAKs and cytokine receptors (Verdier *et al.* 1998; Kamura *et al.* 2004; Piessevaux *et al.* 2008). The SH2 domain of SOCS proteins determines the specificity of the SOCS and CIS proteins for the respective cytokine receptors (Endo *et al.* 1997; Nicholson *et al.* 2000).

So far, SOCS1 and SOCS3 are the most studied SOCS proteins. They both contain a Nterminal kinase inhibitory region (KIR) that is absent in other SOCS proteins (fig. 1). The KIRs of SOCS1 and SOCS3 can directly inhibit JAK tyrosine kinase activity, acting as pseudo-substrates, and by that can block the interaction of JAKs with their substrate STAT molecules. JAK inhibition by SOCS1 and 3 takes place even in the absence of the SOCS box (Yasukawa *et al.* 1999; Zhang *et al.* 2001). SOCS1 can bind to the catalytic domain of JAK2 and to Tyk2 a molecule of the JAK family that mediates IFN- signalling. SOCS1 has also been shown to bind directly to type I IFN receptors (IFNar1) (Fenner *et al.* 2006) and to the IFN- receptor inhibiting efficiently STAT1-mediated signalling (Kubo *et al.* 2003; Qing *et al.* 2005; Fenner *et al.* 2006). Furthermore, SOCS1 can bind to the insulin receptor (Mooney *et al.* 2001; Rui *et al.* 2002) and to the glucocorticoid receptor via its SH2 domain (Haffner *et al.* 2008). Besides JAK2, SOCS3 binds to a variety of receptors as the common IL-6 family coreceptor gp130 at the phosphorylated tyrosine 757, erythropoietin receptor, granulocyte colony stimulating factor (GCSF) receptor, growth hormone receptor (Hansen *et al.* 1999;

Fig. 1. Schematic structure of SOCS1 and SOCS3 proteins shown in upper panel, SOCS1 and SOCS3 as negative regulators of JAK/STAT signalling in lower panel. SOCS1 preferably binds directly to the JAK activation loop while the SH2 domain of SOCS3 binds to the cytokine receptor.

Nicholson *et al.* 2000; Sasaki *et al.* 2000; Hortner *et al.* 2002a; Hortner *et al.* 2002b) and cytokine receptors IL2R and IL-12R1(Cohney *et al.* 1999; Yamamoto *et al.* 2003). Possible interactions of SOCS1 and SOCS3 with Toll-like Receptor (TLR) signalling have been described and will be discussed later.

#### **3. SOCS1**

Inflammation, Chronic Diseases and Cancer –

280 Cell and Molecular Biology, Immunology and Clinical Bases

SOCS proteins play a role in balancing immune functions at different levels, including the differentiation of immune cell populations and their activation by environmental stimuli. Both deletion and over-expression of SOCS proteins in animal models provided insights into their importance of regulating the responsiveness to cytokines. Accumulated today's knowledge on the immunobiology of SOCS proteins convert them into new potential targets for treatment of inflammatory diseases but might also help to improve infection control.

The family of SOCS proteins consists of 8 members (cytokine inducible SH2 protein, CIS, SOCS1-7), which share a central modulator organization with a SH2 domain, a C-terminal SOCS box and an amino-terminal domain of variable length (fig. 1). The SOCS-box was shown to interact with elongin A and B, cullin 5 and ring box. This complex acts as an E3 ubiquitin ligase, initiating ubiquitination in other words the covalent binding of ubiquitin to target proteins, which is followed by the proteosomal degradation of bound signalling complexes as JAKs and cytokine receptors (Verdier *et al.* 1998; Kamura *et al.* 2004; Piessevaux *et al.* 2008). The SH2 domain of SOCS proteins determines the specificity of the SOCS and CIS

So far, SOCS1 and SOCS3 are the most studied SOCS proteins. They both contain a Nterminal kinase inhibitory region (KIR) that is absent in other SOCS proteins (fig. 1). The KIRs of SOCS1 and SOCS3 can directly inhibit JAK tyrosine kinase activity, acting as pseudo-substrates, and by that can block the interaction of JAKs with their substrate STAT molecules. JAK inhibition by SOCS1 and 3 takes place even in the absence of the SOCS box (Yasukawa *et al.* 1999; Zhang *et al.* 2001). SOCS1 can bind to the catalytic domain of JAK2 and to Tyk2 a molecule of the JAK family that mediates IFN- signalling. SOCS1 has also been shown to bind directly to type I IFN receptors (IFNar1) (Fenner *et al.* 2006) and to the IFN- receptor inhibiting efficiently STAT1-mediated signalling (Kubo *et al.* 2003; Qing *et al.* 2005; Fenner *et al.* 2006). Furthermore, SOCS1 can bind to the insulin receptor (Mooney *et al.* 2001; Rui *et al.* 2002) and to the glucocorticoid receptor via its SH2 domain (Haffner *et al.* 2008). Besides JAK2, SOCS3 binds to a variety of receptors as the common IL-6 family coreceptor gp130 at the phosphorylated tyrosine 757, erythropoietin receptor, granulocyte colony stimulating factor (GCSF) receptor, growth hormone receptor (Hansen *et al.* 1999;

Fig. 1. Schematic structure of SOCS1 and SOCS3 proteins shown in upper panel, SOCS1 and SOCS3 as negative regulators of JAK/STAT signalling in lower panel. SOCS1 preferably binds directly to the JAK activation loop while the SH2 domain of SOCS3 binds to the

proteins for the respective cytokine receptors (Endo *et al.* 1997; Nicholson *et al.* 2000).

**2. SOCS at the molecular level**

cytokine receptor.

SOCS1 is expressed constitutively in the thymus, spleen, lung and testes of mice. SOCS1 mRNA expression can be rapidly induced by many cytokines, especially IFNs, and serves as a classical feed back loop inhibiting its inducing pathway (Naka *et al.* 1997; Starr *et al.* 1997). Importantly, SOCS1 mRNA expression increases even in response to microbial molecules such as LPS, Pam3Cys and CpG oligonucleotides that signal via TLR (Dalpke *et al.* 2001; Alexander 2002; Fujimoto & Naka 2003; Dennis *et al.* 2006). Furthermore, hormones like insulin (Emanuelli *et al.* 2000), cardiotrophin (Hamanaka *et al.* 2001) or glucocorticoids (Bhattacharyya *et al.* 2011) have been shown to stimulate SOCS1 expression.

SOCS1-/- animals die within 3 weeks after birth due to fatty degeneration and necrosis of the liver (Naka *et al.* 1998; Starr *et al.* 1998).  At least in part, the spontaneous inflammatory disease is thought to be due to IFN- hyper-responsiveness of SOCS1-deficient tissue, because SOCS1-/-IFN--/- as well as SOCS1-/-STAT1-/- mice survive healthy until adulthood (Alexander *et al.* 1999; Marine *et al.* 1999b). The importance of IFN- was further apparent in mice heterozygous for IFN- and lacking SOCS1 that survived until 5 month of age before succumbing with myocardis and polymyositis (Metcalf *et al.* 2000). SOCS1 mice, treated with neutralizing antibodies from birth, died of the same phenotype reaching adulthood (Bullen *et al.* 2001). A similar expected protection from lethality was observed in SOCS1-/ crossed with IFN-R-/- and STAT1-/- mice. Furthermore, RAG2-/-SOCS1-/- are healthy to at least 3 month of age, implicating that IFN- secreted by T and/ or NKT cells might be responsible for the tremendous inflammation observed (Marine *et al.* 1999b). In fact, depletion of NKT cell by antibody-treatment significantly increased the survival of mice. Surprisingly, mice with a conditional knock down for SOCS1 in T and NKT cells did not display any of the SOCS1-/- pathologies, indicating that a deletion of SOCS1 in T and NKT cells is not sufficient for the hyper-inflammation but that this was due an uncontrolled response of myeloid cells together with an excessive neonatal IFN- release (Chong *et al.* 2003). The fact that myeloid cells are involved was demonstrated by the lethality of chimeric mice, which received SOCS1-/- bone marrow after irradiation (Metcalf *et al.* 2003). On the other hand, T cells were also required for SOCS1-mediated lethality since SOCS1-/- mice with a SOCS1 transgene expressed only by T cells survived. Interestingly, the survival of SOCS1-/- mice also increased when back-crossed to IFNar1-/- mice (lacking one of the IFN- R subunits) indicating that even type I IFNs in part contribute to the lethal inflammation of SOCS1 deficient animals. Altogether, SOCS1 lethality is mainly due to both an exacerbated secretion of IFN- by T and NKT cells during the neonatal period and to a hyper-response of myeloid cells to IFN-, but IFN--independent immune responses such as type I IFN and others discussed below are also likely to contribute to it.

The use of conditional knock down mice, as well as in vitro experiments with different cell populations allowed a more precise definition of the role of SOCS1 in different cell lineages. A combination of SOCS1 conditional knock downs in hematopoetic cells using the cre-lox technology confirmed that SOCS1 deficiency in both T cells and macrophages

"Suppressor of Cytokine Signalling" Molecules in Infection and Inflammation 283

SOCS1 deficient mice are hypersensitive to LPS-induced endotoxic shock, associated with increased levels of IL-12 and TNF- (Kinjyo *et al.* 2002). Surprisingly, even though IFN-R-/ mice are very resistant to endotoxic shock (Car *et al.* 1994), additional knock-out of IFN- or STAT1 did not rescue SOCS1-/- mice from lethal LPS injection, demonstrating that SOCS1 attenuates IFN-independent mechanisms that mediate septic lethal shock (Car *et al.* 1994; Kinjyo *et al.* 2002; Nakagawa *et al.* 2002). Furthermore, elevated sensitivity to endotoxin shock was observed in SOCS1fl/fl LysM-cre mice lacking SOCS1 in macrophages and neutrophils (Hashimoto *et al.* 2009b). Another contributing factor to the elevated sensitivity might be that SOCS1 mediates the protective effect of cardiotrophin-1 in sepsis-induced

These findings suggest an interaction of SOCS1 with components of TLR signalling underlying the role of SOCS1 in protection against septic shock. In fact, binding of SOCS1 to IRAK1 and the p65 subunit of NFB has been shown to destabilize and limit NFB activation (Kinjyo *et al.* 2002; Nakagawa *et al.* 2002; Ryo *et al.* 2003; Maine *et al.* 2007). Others demonstrated that SOCS1 might also bind to apoptosis signal-regulating kinase 1 (ASK1) and regulate mitogen-activated protein kinases JNK and p38 (He *et al.* 2006). Furthermore, SOCS1 was shown to mediate the degradation of the adaptor protein Mal, involved in TLR2 and TLR4 signalling (Mansell *et al.* 2006). However, results from other investigators could not confirm a direct effect of SOCS1 on TLR signalling (Baetz *et al.* 2004; Gingras *et al.* 2004) . SOCS1 over-expression did not affect TLR signalling, instead the inhibition of IFN- mediated STAT1 activation by SOCS1 has been suggested to account for the observed sensitivity to LPS in IFN--/-/SOCS1-/- mice (Baetz *et al.* 2004; Gingras *et al.* 2004). Thus, further work is required to clarify whether SOCS1 is directly involved in the regulation of

A wide range of pathogens including parasites, bacteria and viruses are potent stimulators of SOCS1 expression in the host. Despite obvious differences in the immunobiology and in the type of protective or deleterious immune responses elicited, for most intracellular infections studied, SOCS1 expression is apparently facilitating pathogen replication. But by hampering inflammatory reactions SOCS1 may also improve the pathological outcome of infections and thereby reduce morbidity. In this respect, manipulation of the immune system by SOCS1 conveys an adaptive advantage to pathogens. Microbes replicating in macrophages can be thought of hijacking the host

To unravel the role of SOCS1 different infection models knock out and conditional knock down mouse strains have been applied. Other tools, such as lentiviruses encoding SOCS1 siRNA and treatment with small molecules inhibiting or mimicking SOCS1 action have also been used. Altogether, targeting SOCS1 may serve as a tool to improve the control of

Several viruses including Herpes Simplex Virus (HSV)(Frey *et al.* 2009), human respiratory syncytial virus (RSV) (Hashimoto *et al.* 2009a), hepatitis C (HCV)(Yao *et al.* 2011), Ebola (Okumura *et al.* 2010) and human-immunodeficiency (HIV)(Yadav *et al.* 2009) virus were found to up regulate SOCS1 (Yang *et al.* 2008; Frey *et al.* 2009; Hashimoto *et al.* 2009a;

**3.1 Role of SOCS1 in infectious diseases and inflammation**

cardiomyocyte depression (Tanimoto *et al.* 2005).

SOCS system as an immune evasion mechanism.

different infections and their pathological outcomes.

TLR signalling.

**3.1.1 Viral infections**

is critical to result in lethal inflammation (Chong *et al.* 2005). SOCS1fl/- LysM-cre mice deficient for SOCS1 in myeloid cells showed signs of morbidity starting from 50 days of age with splenomegaly as the most prominent feature. Moribund SOCS1lox/- LysM-cre mice showed aberrantly activated T cells and elevated serum levels of IFN-, TNF-α and IL-12 p40. SOCS1fl/fl Lck-cre mice, deleted for SOCS1 in T- and NKT-cell compartment did not develop lethal multi-organ inflammation but developed multiple lymphoid abnormalities, including enhanced differentiation of thymocytes toward CD8 T cells and very high percentages of peripheral CD8 T cells with a memory phenotype (Chong *et al.* 2005).

SOCS1-/- mice could also be partially protected from neonatal lethality when crossed with STAT1-/-, STAT4-/- or STAT6-/- mice but nevertheless showed a chronic inflammation with aberrant T cell activation (Metcalf *et al.* 2000; Naka *et al.* 2001; Eyles *et al.* 2002). STAT6 and STAT4 are two STAT family members that specifically mediate signals that emanate from the IL-4/ IL-13 and IL-12 receptors, respectively (Wurster *et al.* 2000). IL-4/ IL-13 and IL-12 are cytokines that are important regulators of the proliferation, differentiation and functional capacity of lymphocytes. Although the impact of genetic deletion of STAT4 and STAT6 genes in the survival of SOCS1 deficient animals is lower than that of STAT1, this demonstrates that SOCS1 also regulates IL-4 and IL-12 signalling. Uncontrolled responses to IL-12, an inflammatory cytokine, could contribute to increased IFN- secretion by T cells production and, as indicated above, to the development of inflammatory disease in SOCS1-/ mice (Eyles *et al.* 2002). IL-4, like IFN-, induces de novo expression of SOCS-1 in primary macrophages. Induction of SOCS1 gene expression by IL-4 is STAT6-dependent and SOCS1 feedback inhibits expression of STAT6-responsive genes. Upon binding to their ligand the IL-4 and IL-12 receptors induce tyrosine phosphorylation of the JAK2 protein kinase (Wurster *et al.* 2000). In relation, SOCS1 deletion in T cells resulted in an elevated production of both IFN- and IL-4, which might indicate an enhanced function of Th2 populations in addition to Th1 cells. Overexpression of SOCS1 in Th2 cells has been shown to repress STAT6 activation and inhibit IL-4-induced proliferation, while depletion of SOCS1 by an anti-sense SOCS1 enhanced cell proliferation and induced activation of STAT6 in Th2 cells (Yu *et al.* 2004). On the other hand, the role of SOCS1 in a Th2 skewed immune responses has been suggested (Lee *et al.* 2009), in relation to the fact that SOCS1 expression is 5-fold higher in Th1 than in Th2 cells (Egwuagu *et al.* 2002).

Dendritic cells (DCs) play a critical role in initiating and regulating adaptive immune responses. Silencing of SOCS1 in DCs broke self-tolerance to tumor antigens and increased the magnitude of antigen-presentation (Shen *et al.* 2004; Evel-Kabler *et al.* 2006). Moreover, the increased responses of SOCS1-/- T and NK cells to IL-12 (Eyles *et al.* 2002), and the IFN- mediated inflammatory disease in mice deficient for SOCS1 in regulatory T (Treg) cells (Lu *et al.* 2009; Lu *et al.* 2010) showed a SOCS1-mediated control of Treg cell function, but also highlights the relevance of SOCS1 as target molecule in these diseases. In Treg cells specific microRNA regulated SOCS1 expression.

SOCS1 has also been shown to inhibit B cell proliferation in vitro and autoantibody production in vivo, through suppression of BAFF/BLyS a B cell growth and differentiation factor, that has been implicated in systemic autoimmune diseases (Hanada *et al.* 2003).

Taken together, several findings indicate that SOCS1 is involved in the regulation of Th1 responses by modulating the responses of different immune cells, such as macrophages, DCs, Th1 and Treg cells.

#### **3.1 Role of SOCS1 in infectious diseases and inflammation**

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282 Cell and Molecular Biology, Immunology and Clinical Bases

is critical to result in lethal inflammation (Chong *et al.* 2005). SOCS1fl/- LysM-cre mice deficient for SOCS1 in myeloid cells showed signs of morbidity starting from 50 days of age with splenomegaly as the most prominent feature. Moribund SOCS1lox/- LysM-cre mice showed aberrantly activated T cells and elevated serum levels of IFN-, TNF-α and IL-12 p40. SOCS1fl/fl Lck-cre mice, deleted for SOCS1 in T- and NKT-cell compartment did not develop lethal multi-organ inflammation but developed multiple lymphoid abnormalities, including enhanced differentiation of thymocytes toward CD8 T cells and very high percentages of peripheral CD8 T cells with a memory phenotype (Chong *et al.*

SOCS1-/- mice could also be partially protected from neonatal lethality when crossed with STAT1-/-, STAT4-/- or STAT6-/- mice but nevertheless showed a chronic inflammation with aberrant T cell activation (Metcalf *et al.* 2000; Naka *et al.* 2001; Eyles *et al.* 2002). STAT6 and STAT4 are two STAT family members that specifically mediate signals that emanate from the IL-4/ IL-13 and IL-12 receptors, respectively (Wurster *et al.* 2000). IL-4/ IL-13 and IL-12 are cytokines that are important regulators of the proliferation, differentiation and functional capacity of lymphocytes. Although the impact of genetic deletion of STAT4 and STAT6 genes in the survival of SOCS1 deficient animals is lower than that of STAT1, this demonstrates that SOCS1 also regulates IL-4 and IL-12 signalling. Uncontrolled responses to IL-12, an inflammatory cytokine, could contribute to increased IFN- secretion by T cells production and, as indicated above, to the development of inflammatory disease in SOCS1-/ mice (Eyles *et al.* 2002). IL-4, like IFN-, induces de novo expression of SOCS-1 in primary macrophages. Induction of SOCS1 gene expression by IL-4 is STAT6-dependent and SOCS1 feedback inhibits expression of STAT6-responsive genes. Upon binding to their ligand the IL-4 and IL-12 receptors induce tyrosine phosphorylation of the JAK2 protein kinase (Wurster *et al.* 2000). In relation, SOCS1 deletion in T cells resulted in an elevated production of both IFN- and IL-4, which might indicate an enhanced function of Th2 populations in addition to Th1 cells. Overexpression of SOCS1 in Th2 cells has been shown to repress STAT6 activation and inhibit IL-4-induced proliferation, while depletion of SOCS1 by an anti-sense SOCS1 enhanced cell proliferation and induced activation of STAT6 in Th2 cells (Yu *et al.* 2004). On the other hand, the role of SOCS1 in a Th2 skewed immune responses has been suggested (Lee *et al.* 2009), in relation to the fact that SOCS1 expression is 5-fold

Dendritic cells (DCs) play a critical role in initiating and regulating adaptive immune responses. Silencing of SOCS1 in DCs broke self-tolerance to tumor antigens and increased the magnitude of antigen-presentation (Shen *et al.* 2004; Evel-Kabler *et al.* 2006). Moreover, the increased responses of SOCS1-/- T and NK cells to IL-12 (Eyles *et al.* 2002), and the IFN- mediated inflammatory disease in mice deficient for SOCS1 in regulatory T (Treg) cells (Lu *et al.* 2009; Lu *et al.* 2010) showed a SOCS1-mediated control of Treg cell function, but also highlights the relevance of SOCS1 as target molecule in these diseases. In Treg cells specific

SOCS1 has also been shown to inhibit B cell proliferation in vitro and autoantibody production in vivo, through suppression of BAFF/BLyS a B cell growth and differentiation factor, that has been implicated in systemic autoimmune diseases (Hanada

Taken together, several findings indicate that SOCS1 is involved in the regulation of Th1 responses by modulating the responses of different immune cells, such as macrophages,

higher in Th1 than in Th2 cells (Egwuagu *et al.* 2002).

microRNA regulated SOCS1 expression.

*et al.* 2003).

DCs, Th1 and Treg cells.

2005).

SOCS1 deficient mice are hypersensitive to LPS-induced endotoxic shock, associated with increased levels of IL-12 and TNF- (Kinjyo *et al.* 2002). Surprisingly, even though IFN-R-/ mice are very resistant to endotoxic shock (Car *et al.* 1994), additional knock-out of IFN- or STAT1 did not rescue SOCS1-/- mice from lethal LPS injection, demonstrating that SOCS1 attenuates IFN-independent mechanisms that mediate septic lethal shock (Car *et al.* 1994; Kinjyo *et al.* 2002; Nakagawa *et al.* 2002). Furthermore, elevated sensitivity to endotoxin shock was observed in SOCS1fl/fl LysM-cre mice lacking SOCS1 in macrophages and neutrophils (Hashimoto *et al.* 2009b). Another contributing factor to the elevated sensitivity might be that SOCS1 mediates the protective effect of cardiotrophin-1 in sepsis-induced cardiomyocyte depression (Tanimoto *et al.* 2005).

These findings suggest an interaction of SOCS1 with components of TLR signalling underlying the role of SOCS1 in protection against septic shock. In fact, binding of SOCS1 to IRAK1 and the p65 subunit of NFB has been shown to destabilize and limit NFB activation (Kinjyo *et al.* 2002; Nakagawa *et al.* 2002; Ryo *et al.* 2003; Maine *et al.* 2007). Others demonstrated that SOCS1 might also bind to apoptosis signal-regulating kinase 1 (ASK1) and regulate mitogen-activated protein kinases JNK and p38 (He *et al.* 2006). Furthermore, SOCS1 was shown to mediate the degradation of the adaptor protein Mal, involved in TLR2 and TLR4 signalling (Mansell *et al.* 2006). However, results from other investigators could not confirm a direct effect of SOCS1 on TLR signalling (Baetz *et al.* 2004; Gingras *et al.* 2004) . SOCS1 over-expression did not affect TLR signalling, instead the inhibition of IFN- mediated STAT1 activation by SOCS1 has been suggested to account for the observed sensitivity to LPS in IFN--/-/SOCS1-/- mice (Baetz *et al.* 2004; Gingras *et al.* 2004). Thus, further work is required to clarify whether SOCS1 is directly involved in the regulation of TLR signalling.

A wide range of pathogens including parasites, bacteria and viruses are potent stimulators of SOCS1 expression in the host. Despite obvious differences in the immunobiology and in the type of protective or deleterious immune responses elicited, for most intracellular infections studied, SOCS1 expression is apparently facilitating pathogen replication. But by hampering inflammatory reactions SOCS1 may also improve the pathological outcome of infections and thereby reduce morbidity. In this respect, manipulation of the immune system by SOCS1 conveys an adaptive advantage to pathogens. Microbes replicating in macrophages can be thought of hijacking the host SOCS system as an immune evasion mechanism.

To unravel the role of SOCS1 different infection models knock out and conditional knock down mouse strains have been applied. Other tools, such as lentiviruses encoding SOCS1 siRNA and treatment with small molecules inhibiting or mimicking SOCS1 action have also been used. Altogether, targeting SOCS1 may serve as a tool to improve the control of different infections and their pathological outcomes.

#### **3.1.1 Viral infections**

Several viruses including Herpes Simplex Virus (HSV)(Frey *et al.* 2009), human respiratory syncytial virus (RSV) (Hashimoto *et al.* 2009a), hepatitis C (HCV)(Yao *et al.* 2011), Ebola (Okumura *et al.* 2010) and human-immunodeficiency (HIV)(Yadav *et al.* 2009) virus were found to up regulate SOCS1 (Yang *et al.* 2008; Frey *et al.* 2009; Hashimoto *et al.* 2009a;

"Suppressor of Cytokine Signalling" Molecules in Infection and Inflammation 285

improving production and stability of HIV-1 particles (Ryo et al. 2008). In a HIV vaccination model in mice, silencing of SOCS1 in DC using siRNA increased the secretion of IL-12 and other pro-inflammatory cytokines resulting in enhanced memory humoral and cellular immune responses (Song *et al.* 2006). Recently, it was shown that treatment of DCs from HIV patients with SOCS1 siRNA augmented the frequency of polyfunctional cytotoxic T cells (T cells secreting IL-2, TNF- and IFN-are associated with protection)(Subramanya *et al.* 2010), suggesting that silencing SOCS1 in DCs may

Taken together, SOCS1 induction is a successful tool of various viruses to diminish antiviral

Studies on the role of SOCS1 have been focussed on intracellular bacterial infections in which IFN- usually plays a major protective role. Research from our laboratory showed that *Chlamydia pneumoniae* infection of macrophages induced SOCS1 expression in a STAT-1 and IFN-dependent manner (Yang *et al.* 2008). Infected SOCS1-/- macrophages displayed lower bacterial titers and higher levels of IFN-regulated genes as iNOS (inducible nitric oxide synthase) and IDO (indoleamine dioxygenase), that participate in the control of intracellular bacteria. RAG1-/-/SOCS1-/- mice showed 10-fold lower bacteria numbers in lungs than controls 6 days after infection. However, RAG1-/-/SOCS1-/- mice died within seven days after infection with *C. pneumoniae* showing a severe pulmonary inflammation, whereas RAG1-/- mice survived for more than 60 days. Thus, SOCS1 has a crucial role in

Tuberculosis is another disease in which IFN- plays a critical role in containing bacterial levels and in the maintenance of the latent asymptomatic disease in infected individuals. Infection with different mycobacterial species such as *Mycobacterium bovis* (Imai *et al.* 2003), *M. avium* (Vazquez *et al.* 2006) and *M. tuberculosis* have been shown to stimulate SOCS1 in macrophages (Imai *et al.* 2003; Vazquez *et al.* 2006; Srivastava *et al.* 2009; Carow *et al.* 2011). Different innate immune receptors including TLR, NLR and DC-SIGN were found to mediate SOCS1 stimulation in mycobacterial infected myeloid cells (Srivastava

Experiments from our laboratory and others, showed improved *M. tuberculosis* control in SOCS1 deficient macrophages and DCs (Srivastava *et al.* 2009; Carow *et al.* 2011). SOCS1-/ infected macrophages expressed higher levels of IFN--responsive genes but also higher levels of IFN- itself than infected control cells. In agreement, SOCS1-/- macrophages secreted higher amounts of IFN- in response to IL-12, and although levels of IL-12 were not altered, SOCS1-/- macrophages displayed increased IL-12R1 expression (Fig. 2). Of importance, the improved control of *M. tuberculosis* or *M. bovis* BCG by SOCS1-/- was lost in IFN--/-/SOCS1-/- macrophages, indicating that IFN- secretion mediates the improved mycobacterial control by SOCS1-/- macrophages. Moreover, despite SOCS1 expression, *M. tuberculosis*-infected macrophages were unimpaired in their response to IFN-. Altogether, as depicted in fig. 2, this suggests that SOCS1 regulates the secretion of rather than the response to IFN- via controlling responses to IL-12 and that this causes mycobacterial resistance of macrophages. As stated above, SOCS1 regulates STAT4 activation in response

to IL-12, which probably accounts for this mechanism (Eyles *et al.* 2002) (Fig. 2).

IFN responses, leading in many of the infections to impaired control of infection.

contribute to improved HIV-vaccination.

preventing lethal inflammation in *C. pneumoniae* infection.

**3.1.2 Bacterial infections**

*et al.* 2009).

Hashimoto *et al.* 2009b; Okumura *et al.* 2010; Yao *et al.* 2011). Since SOCS1 is interfering with the type I IFN signalling, a role for SOCS1 in incrementing susceptibility to viral infection could be expected.

SOCS1-/-/IFN--/- mice outlived Semliki Forest virus (SFV)-infected control mice substantially (Alexander *et al.* 1999). Even though serum levels of IFN- were lower in infected SOCS1-/- mice than in controls, an increased sensitivity to IFN- was associated to resistance. The IFN-ar1 chain of the IFN- receptor was shown to interact with SOCS1 and therefore inhibited efficient host responses (Fenner *et al.* 2006).

In another model, human T cell lymphotropic virus (HTLV) replication in peripheral blood mononuclear cells correlated with induction of SOCS1 and inhibition of IFN- and IFNstimulated gene expression (Oliere *et al.* 2010). These authors also showed that HTLV infection-induced SOCS1 mediated proteosomal degradation of IRF3, the blockade of IFNgene expression and enhanced viral load. Of importance, SOCS1 did not only inhibit type I IFN-mediated viral defences but was found to impair efficient IFN- stimulated antitumoral defences in mice (Zitzmann *et al.* 2007).

*In vivo*, injection of dnSOCS1 construct into the hearts of coxsackievirus-infected mice attenuated both virus replication and cardiomyocyte damage, protecting the heart from viral infection, suggesting that SOCS1 is a relevant therapeutic target (Yasukawa *et al.* 2003b).

Recently, specific microRNAs were found to regulate IFN- responses. Infection with vesicular stromatitis virus (VSV), was shown to stimulate miR-155 expression that, by inhibition of SOCS1, positively regulated host antiviral innate immune response by promoting IFN- responses (Wang *et al.* 2010). Thus, while SOCS1 is up-regulated during inflammatory IFN-mediated responses, miRNA might be a new mechanism by which the host can fine-tune its antiviral state.

Besides type I IFNs, responses to IFN- can be an essential component of control of some viral infections. The HSV-infection in keratinocytes, stimulated SOCS1 expression and made them refractory to IFN-, whereas HSV infected fibroblasts did not increase SOCS1 levels and developed an antiviral state after IFN- stimulation (Frey *et al.* 2009). Treatment of cells with a SOCS1 antagonist peptide as well as silencing SOCS1 with siRNA protected keratinocytes from HSV-1 infection and restored responsiveness to IFN-

In contrast to suppression of IFN-mediated protective responses by SOCS1, in other virus infections unrestrained immune responses can cause damaging immunopathology. In these cases impairment of cytokine responses with SOCS1 may prevent inflammatory damage. Thus, in a vaccinia virus infection model, the administration of a small tyrosine inhibitor peptide that binds to JAK2 and inhibits STAT1 phosphorylation as well as a peptide mimicking the SOCS1 KIR region protected mice against lethal virus infection (Ahmed *et al.* 2009).

Clinical studies showed T cell exhaustion in chronically HCV-infected individuals. Interestingly, the expression of programmed death receptor 1 (PD1) and SOCS1 was increased in T cells and macrophages (Frazier *et al.* 2010; Zhang *et al.* 2011). In both cell types, blocking of PD1 signalling reduced SOCS1 expression and led to improved T cell proliferation and IL-12 secretion by macrophages respectively.

Studies on HIV progressor patients showed that SOCS1 expression was elevated in CD4 T cells displaying reduced IRF1 expression after IFN- stimulation in comparison to healthy controls (Yadav *et al.* 2009). Furthermore, SOCS1 was found to bind to HIV gag protein improving production and stability of HIV-1 particles (Ryo et al. 2008). In a HIV vaccination model in mice, silencing of SOCS1 in DC using siRNA increased the secretion of IL-12 and other pro-inflammatory cytokines resulting in enhanced memory humoral and cellular immune responses (Song *et al.* 2006). Recently, it was shown that treatment of DCs from HIV patients with SOCS1 siRNA augmented the frequency of polyfunctional cytotoxic T cells (T cells secreting IL-2, TNF- and IFN-are associated with protection)(Subramanya *et al.* 2010), suggesting that silencing SOCS1 in DCs may contribute to improved HIV-vaccination.

Taken together, SOCS1 induction is a successful tool of various viruses to diminish antiviral IFN responses, leading in many of the infections to impaired control of infection.

#### **3.1.2 Bacterial infections**

Inflammation, Chronic Diseases and Cancer –

284 Cell and Molecular Biology, Immunology and Clinical Bases

Hashimoto *et al.* 2009b; Okumura *et al.* 2010; Yao *et al.* 2011). Since SOCS1 is interfering with the type I IFN signalling, a role for SOCS1 in incrementing susceptibility to viral infection

SOCS1-/-/IFN--/- mice outlived Semliki Forest virus (SFV)-infected control mice substantially (Alexander *et al.* 1999). Even though serum levels of IFN- were lower in infected SOCS1-/- mice than in controls, an increased sensitivity to IFN- was associated to resistance. The IFN-ar1 chain of the IFN- receptor was shown to interact with SOCS1 and

In another model, human T cell lymphotropic virus (HTLV) replication in peripheral blood mononuclear cells correlated with induction of SOCS1 and inhibition of IFN- and IFNstimulated gene expression (Oliere *et al.* 2010). These authors also showed that HTLV infection-induced SOCS1 mediated proteosomal degradation of IRF3, the blockade of IFNgene expression and enhanced viral load. Of importance, SOCS1 did not only inhibit type I IFN-mediated viral defences but was found to impair efficient IFN- stimulated anti-

*In vivo*, injection of dnSOCS1 construct into the hearts of coxsackievirus-infected mice attenuated both virus replication and cardiomyocyte damage, protecting the heart from viral infection, suggesting that SOCS1 is a relevant therapeutic target (Yasukawa *et al.*

Recently, specific microRNAs were found to regulate IFN- responses. Infection with vesicular stromatitis virus (VSV), was shown to stimulate miR-155 expression that, by inhibition of SOCS1, positively regulated host antiviral innate immune response by promoting IFN- responses (Wang *et al.* 2010). Thus, while SOCS1 is up-regulated during inflammatory IFN-mediated responses, miRNA might be a new mechanism by which the

Besides type I IFNs, responses to IFN- can be an essential component of control of some viral infections. The HSV-infection in keratinocytes, stimulated SOCS1 expression and made them refractory to IFN-, whereas HSV infected fibroblasts did not increase SOCS1 levels and developed an antiviral state after IFN- stimulation (Frey *et al.* 2009). Treatment of cells with a SOCS1 antagonist peptide as well as silencing SOCS1 with siRNA protected

In contrast to suppression of IFN-mediated protective responses by SOCS1, in other virus infections unrestrained immune responses can cause damaging immunopathology. In these cases impairment of cytokine responses with SOCS1 may prevent inflammatory damage. Thus, in a vaccinia virus infection model, the administration of a small tyrosine inhibitor peptide that binds to JAK2 and inhibits STAT1 phosphorylation as well as a peptide mimicking the SOCS1 KIR region protected mice against lethal virus infection

Clinical studies showed T cell exhaustion in chronically HCV-infected individuals. Interestingly, the expression of programmed death receptor 1 (PD1) and SOCS1 was increased in T cells and macrophages (Frazier *et al.* 2010; Zhang *et al.* 2011). In both cell types, blocking of PD1 signalling reduced SOCS1 expression and led to improved T cell

Studies on HIV progressor patients showed that SOCS1 expression was elevated in CD4 T cells displaying reduced IRF1 expression after IFN- stimulation in comparison to healthy controls (Yadav *et al.* 2009). Furthermore, SOCS1 was found to bind to HIV gag protein

keratinocytes from HSV-1 infection and restored responsiveness to IFN-

proliferation and IL-12 secretion by macrophages respectively.

therefore inhibited efficient host responses (Fenner *et al.* 2006).

tumoral defences in mice (Zitzmann *et al.* 2007).

host can fine-tune its antiviral state.

(Ahmed *et al.* 2009).

could be expected.

2003b).

Studies on the role of SOCS1 have been focussed on intracellular bacterial infections in which IFN- usually plays a major protective role. Research from our laboratory showed that *Chlamydia pneumoniae* infection of macrophages induced SOCS1 expression in a STAT-1 and IFN-dependent manner (Yang *et al.* 2008). Infected SOCS1-/- macrophages displayed lower bacterial titers and higher levels of IFN-regulated genes as iNOS (inducible nitric oxide synthase) and IDO (indoleamine dioxygenase), that participate in the control of intracellular bacteria. RAG1-/-/SOCS1-/- mice showed 10-fold lower bacteria numbers in lungs than controls 6 days after infection. However, RAG1-/-/SOCS1-/- mice died within seven days after infection with *C. pneumoniae* showing a severe pulmonary inflammation, whereas RAG1-/- mice survived for more than 60 days. Thus, SOCS1 has a crucial role in preventing lethal inflammation in *C. pneumoniae* infection.

Tuberculosis is another disease in which IFN- plays a critical role in containing bacterial levels and in the maintenance of the latent asymptomatic disease in infected individuals. Infection with different mycobacterial species such as *Mycobacterium bovis* (Imai *et al.* 2003), *M. avium* (Vazquez *et al.* 2006) and *M. tuberculosis* have been shown to stimulate SOCS1 in macrophages (Imai *et al.* 2003; Vazquez *et al.* 2006; Srivastava *et al.* 2009; Carow *et al.* 2011). Different innate immune receptors including TLR, NLR and DC-SIGN were found to mediate SOCS1 stimulation in mycobacterial infected myeloid cells (Srivastava *et al.* 2009).

Experiments from our laboratory and others, showed improved *M. tuberculosis* control in SOCS1 deficient macrophages and DCs (Srivastava *et al.* 2009; Carow *et al.* 2011). SOCS1-/ infected macrophages expressed higher levels of IFN--responsive genes but also higher levels of IFN- itself than infected control cells. In agreement, SOCS1-/- macrophages secreted higher amounts of IFN- in response to IL-12, and although levels of IL-12 were not altered, SOCS1-/- macrophages displayed increased IL-12R1 expression (Fig. 2). Of importance, the improved control of *M. tuberculosis* or *M. bovis* BCG by SOCS1-/- was lost in IFN--/-/SOCS1-/- macrophages, indicating that IFN- secretion mediates the improved mycobacterial control by SOCS1-/- macrophages. Moreover, despite SOCS1 expression, *M. tuberculosis*-infected macrophages were unimpaired in their response to IFN-. Altogether, as depicted in fig. 2, this suggests that SOCS1 regulates the secretion of rather than the response to IFN- via controlling responses to IL-12 and that this causes mycobacterial resistance of macrophages. As stated above, SOCS1 regulates STAT4 activation in response to IL-12, which probably accounts for this mechanism (Eyles *et al.* 2002) (Fig. 2).

"Suppressor of Cytokine Signalling" Molecules in Infection and Inflammation 287

the type of the infection and the genetics of the host whether SOCS1 improves or worsens

Fig. 3. Role of SOCS1 during different stages of M. tuberculosis infection

2003b). However, the underlying mechanisms were not revealed.

but without reduction of parasites (Bullen *et al.* 2003a).

genes after *T. gondii* infection (Zimmermann *et al.* 2006).

The role of SOCS1 was also studied in a limited number of infections with parasites. SOCS1- /- mice were resistant to development of cerebral malaria after infection with *Plasmodium berghei*. This is surprising since IFN- mediates cerebral malaria in this model (Bullen *et al.*

SOCS1-/- macrophages were capable of killing *Leishmania major* at a 100-fold lower IFN concentration than WT macrophages (Alexander *et al.* 1999). However, SOCS1+/- *L. major*infected mice showed a worsened infection immunopathology, with larger dermal lesions

Another parasite, *Toxoplasma gondii* induced *SOCS1* expression in macrophages. SOCS1 over-expressing macrophages were unable to control parasite growth in response to IFN whereas SOCS1-/- macrophages were restored in their ability to induce IFN- responsive

Moreover, recent data show that implantation of the parasite *Brugia malayi*, a causative agent of lymphatic filariasis, generated Th2 responses associated with development of M2 macrophages in mice. Expression of SOCS1 not only controlled the secretion of proinflammatory cytokines in M1 macrophages but was involved in maintaining M2 differentiation (Whyte *et al.* 2011). In line, patients with filariasis showed increased Th2 and impaired Th1 antigen-specific responses, which was associated with increased T cell expression by SOCS1 (Babu *et al.* 2005). Altogether, the function of SOCS1 during infections with parasites resembles that in bacterial infections, balancing anti-pathogen responses and

SOCS3 is an important endogenous inhibitor of STAT3-mediated cytokine signalling. SOCS3 has been shown to be induced by different hormones including ciliary neutrophilic factor,

the outcome of infection.

**3.1.3 Parasite infections**

the severity of inflammation.

**4. SOCS3**

Fig. 2. SOCS1 in *M. tuberculosis*-infected macrophages

Macrophage responses and control of *M. tuberculosis* infection in absence (left panel) or presence (right panel) of SOCS1 regulating IFN- mediated genes.

RAG1-/-SOCS1-/- as well as SOCS1fl/fl LysM-cre mice displayed lower bacterial loads in the lungs early after aerosol infection with *M. tuberculosis*, indicating that SOCS1 expression by macrophages facilitates *M. tuberculosis* infection. However, RAG1-/-/SOCS1-/- and IFN--/- /SOCS1-/- mice showed a dramatic pulmonary inflammation from 3 weeks after *M. tuberculosis* infection without reduced bacteria numbers in different organs. This inflammatory response was caused by SOCS1-deficiency in non-macrophage cells, since it was not observed in *M. tuberculosis*-infected SOCS1fl/fl LysM-cre mice. Overall, *M. tuberculosis* infection induces SOCS1 that by diminishing IL-12 responses impairs IFN secretion by macrophages. This results in lower levels of IFN--regulated genes and in increased pulmonary bacterial levels at early time points. Later during infection, SOCS1 in non-macrophages cells protects mice from severe inflammation. At these later time points, macrophages containing bacteria are able to respond to IFN- and reduce bacterial levels, despite SOCS1 expression. Thus, SOCS1 does not mediate resistance to infection at late time points (fig. 3).

A role of SOCS1 in T cells during *M. tuberculosis* has been envisaged (Srivastava *et al.* 2011). However, SOCS1fl/fl Lck-cre mice deficient for SOCS1 in T cells showed no reduction in *M. tuberculosis* numbers in the lung (unpublished observation).

We have also analysed the expression of SOCS1 in tuberculosis patients. We found higher SOCS1 mRNA levels in blood samples from pulmonary tuberculosis patients than in endemic controls (Srivastava *et al.* 2009; Masood *et al.* submitted ). Furthermore, SOCS1 transcripts were raised in T cells of patients with far advanced as compared with those showing a moderately advanced disease (Masood *et al.* submitted ). This confirmed studies describing an over-representation of SOCS1 (as well as other interferon-induced genes) in active tuberculosis patients compared to latent patients (Berry *et al.* 2010).

In summary, SOCS1 possesses two roles during infection: on the one hand it suppresses protective immune responses but on the other hand also prevents the development of detrimental inflammation. Therefore, it clearly depends on the stage of infection, but also on the type of the infection and the genetics of the host whether SOCS1 improves or worsens the outcome of infection.

Fig. 3. Role of SOCS1 during different stages of M. tuberculosis infection

#### **3.1.3 Parasite infections**

Inflammation, Chronic Diseases and Cancer –

286 Cell and Molecular Biology, Immunology and Clinical Bases

Macrophage responses and control of *M. tuberculosis* infection in absence (left panel) or

RAG1-/-SOCS1-/- as well as SOCS1fl/fl LysM-cre mice displayed lower bacterial loads in the lungs early after aerosol infection with *M. tuberculosis*, indicating that SOCS1 expression by macrophages facilitates *M. tuberculosis* infection. However, RAG1-/-/SOCS1-/- and IFN--/- /SOCS1-/- mice showed a dramatic pulmonary inflammation from 3 weeks after *M. tuberculosis* infection without reduced bacteria numbers in different organs. This inflammatory response was caused by SOCS1-deficiency in non-macrophage cells, since it was not observed in *M. tuberculosis*-infected SOCS1fl/fl LysM-cre mice. Overall, *M. tuberculosis* infection induces SOCS1 that by diminishing IL-12 responses impairs IFN secretion by macrophages. This results in lower levels of IFN--regulated genes and in increased pulmonary bacterial levels at early time points. Later during infection, SOCS1 in non-macrophages cells protects mice from severe inflammation. At these later time points, macrophages containing bacteria are able to respond to IFN- and reduce bacterial levels, despite SOCS1 expression. Thus, SOCS1 does not mediate resistance to infection at late time

A role of SOCS1 in T cells during *M. tuberculosis* has been envisaged (Srivastava *et al.* 2011). However, SOCS1fl/fl Lck-cre mice deficient for SOCS1 in T cells showed no reduction in *M.*

We have also analysed the expression of SOCS1 in tuberculosis patients. We found higher SOCS1 mRNA levels in blood samples from pulmonary tuberculosis patients than in endemic controls (Srivastava *et al.* 2009; Masood *et al.* submitted ). Furthermore, SOCS1 transcripts were raised in T cells of patients with far advanced as compared with those showing a moderately advanced disease (Masood *et al.* submitted ). This confirmed studies describing an over-representation of SOCS1 (as well as other interferon-induced genes) in

In summary, SOCS1 possesses two roles during infection: on the one hand it suppresses protective immune responses but on the other hand also prevents the development of detrimental inflammation. Therefore, it clearly depends on the stage of infection, but also on

Fig. 2. SOCS1 in *M. tuberculosis*-infected macrophages

points (fig. 3).