presence (right panel) of SOCS1 regulating IFN- mediated genes.

*tuberculosis* numbers in the lung (unpublished observation).

active tuberculosis patients compared to latent patients (Berry *et al.* 2010).

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

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

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

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 the severity of inflammation.

#### **4. SOCS3**

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,

"Suppressor of Cytokine Signalling" Molecules in Infection and Inflammation 289

Mice deficient for SOCS3 in myeloid cells are resistant to LPS-induced endotoxic shock whereas STAT3 deficient mice are highly sensitive (Takeda *et al.* 1999; Yasukawa *et al.* 2003a). Thus, STAT3 activation mediates anti-inflammatory IL-10 signalling and in the absence of SOCS3, similar anti-inflammatory properties in response to IL-6 (Johnston & O'Shea 2003). Furthermore, macrophages lacking SOCS3 were shown to secrete reduced

In a similar manner, DCs deficient for STAT3 contributed to T cell hyper-activation while in the absence of SOCS3, DCs induced a selective expansion of regulatory T cells (Cheng *et al.* 2003; Matsumura *et al.* 2007). An overexpression of SOCS3 in DCs resulted a reduction of expression of co-stimulatory molecules and IL-12. Moreover, SOCS3-deficient and overexpressing DCs had the capacity to suppress EAE development (Li *et al.* 2006;

Croker et al. (2003) and Lang et al (2003) on the other hand, using SOCS3 deficient macrophages found an increased STAT1 activation and elevated levels of IFN--regulated

Fig. 4. SOCS3 inhibits anti-inflammatory and IFN-inducible genes in response to IL-6.

Other molecular targets of SOCS3 have been reported: SOCS3 inhibited the activation of TNF-receptor-associated factor 6 (TRAF6) and TGF--activated kinase 1 (TAK1), essential for both TLR– and IL-1-induced responses (Frobose *et al.* 2006). Prele et al. (2006) found no differences in LPS responses of SOCS3 transfected human macrophages. Thus, further studies are required to determine whether there is a direct role for SOCS3 in TLR

Upon over-expression, SOCS3 was found to bind to the GCSF receptor and reduced STAT3 activation in response to GCSF (Hortner *et al.* 2002b; Hermans *et al.* 2003). In agreement, deletion of SOCS3 in the myeloid or hematopoietic cells increased numbers of neutrophils, which showed increased survival and proliferative capacity (Croker *et al.* 2004; Kimura *et al.* 2004). Following GCSF injection SOCS3-deficient mice developed neutrophilia, and a spectrum of inflammatory pathologies, characterised by neutrophil infiltration in multiples tissues (Croker *et al.* 2008). Overall, SOCS3 regulates survival, growth and activation of neutrophils and an inhibition of SOCS3 may enhance the essential neutrophil recovery after

levels of TNF- and IL-12 after IL-6 and LPS stimulation (Yasukawa *et al.* 2003a).

Matsumura *et al.* 2007).

genes in response to IL-6.

Adapted from (Johnston & O'Shea 2003)

chemotherapy or neutropenia.

signalling.

leptin, prolactin and growth hormones (Adams *et al.* 1998; Bjorbaek *et al.* 1998; Bjorbaek *et al.* 1999; Pezet *et al.* 1999) and cytokines like leukemia inhibitory factor, IL-11, IL-10, IL-2, IL-27 and IL-6 (Starr *et al.* 1997; Auernhammer & Melmed 1999; Bousquet *et al.* 1999; Cassatella *et al.* 1999; Cohney *et al.* 1999) but also by microbial molecular patterns as LPS or CpG (Stoiber *et al.* 1999; Dalpke *et al.* 2001).

Among these, the role SOCS3 in regulation of IL-6 responses has been studied in detail. IL-6 is a pro-inflammatory cytokine that has been found to play a role in many inflammatory diseases, while IL-10 is a potent anti-inflammatory cytokine, which suppresses gene activation through TLR signalling pathways, but can also inhibit Th1 responses at different levels. While it is known that STAT3 is essential for the biological actions of both IL-6 and IL-10, it was unclear for many years how these two cytokines could have exactly opposing functions.

Interestingly, an inhibitory role for SOCS3 could only be shown for cytokines binding to the IL-6 receptor whereas the signalling of IL-10 that also stimulates STAT3-activation was unaffected by SOCS3 (Song & Shuai 1998; Lang *et al.* 2003; Yasukawa *et al.* 2003a; Kimura *et al.* 2004; Shuai 2006). This is explained by the ability of SOCS3 to bind to the IL-6 receptor subunit gp130 (Tyr 759) but not to the IL-10 receptor (Nicholson *et al.* 2000; Lehmann *et al.* 2003; Yasukawa *et al.* 2003a). STAT3 activation in response to IL-6 is prolonged in absence of SOCS3. It has been proposed that the sustained activation of STAT3 is essential for the antiinflammatory effect, while transient activation of STAT3 promotes inflammation (Yasukawa *et al.* 2003a).

Gp130 is a promiscuous cytokine receptor subunit that mediates signalling by IL-6 and other cytokines such as IL-11, IL-27, leukemia inhibitory factor (LIF) and cardiotrophin-1 (Taga & Kishimoto 1997). Gp130 is present on hematopoietic and nonhematopoietic cells, and its expression can vary depending on the cell's activation status (Andersson *et al.* 1978). Gp130 itself does not bind to cytokines but acts as a co-receptor. When the cytokine (for example IL-6) binds to the IL-6Ra, it triggers a heterodimeric association with gp130 to form a signalling complex (Silver & Hunter 2010). SOCS3 also modulates signalling of gp-130 dependent cytokines. Moreover, the mutation of SOCS3 binding site on the gp130 receptor increased STAT3 activation in response to IL-6 and stimulated IL-10-like anti-inflammatory responses (Ohtani *et al.* 2000; Croker *et al.* 2003; Lang *et al.* 2003; Yasukawa *et al.* 2003a). Such anti-inflammatory responses are abolished when the gp130 Y759 mutated mice possessed a monoallelic deletion of STAT3 (McLoughlin *et al.* 2005; Yoshimura *et al.* 2007). As already mentioned above, SOCS3 inhibits also signalling via non gp130 containing cytokine and hormone receptors such as GCSF, leptin, IL-12 and even IFNs (Song & Shuai 1998; Bousquet *et al.* 1999; Bjorbak *et al.* 2000; Shen *et al.* 2000; Hortner *et al.* 2002b).

 The early death is caused by enhanced LIF signalling, since SOCS3-/- mice showed altered trophoblast differentiation. Animals, in which the SOCS3 deletion in the placenta was rescued by a tetraploid rescue method, showed extended embryonic life. However, rescued SOCS3-/- embryos died due to cardiac hypertrophy (Takahashi *et al.* 2003).

#### **4.1 SOCS3 in myeloid cells/granulopoiesis**

Mice with a conditional knock down of SOCS3 in myeloid cells demonstrated the role of SOCS3 in suppression of IL-6/gp130 signalling. SOCS3-deficient macrophages stimulated with IL-6 displayed an increased magnitude and duration of STAT1 and STAT3 activation in comparison to controls (Croker *et al.* 2003; Lang *et al.* 2003; Yasukawa *et al.* 2003a).

288 Cell and Molecular Biology, Immunology and Clinical Bases

leptin, prolactin and growth hormones (Adams *et al.* 1998; Bjorbaek *et al.* 1998; Bjorbaek *et al.* 1999; Pezet *et al.* 1999) and cytokines like leukemia inhibitory factor, IL-11, IL-10, IL-2, IL-27 and IL-6 (Starr *et al.* 1997; Auernhammer & Melmed 1999; Bousquet *et al.* 1999; Cassatella *et al.* 1999; Cohney *et al.* 1999) but also by microbial molecular patterns as LPS or CpG (Stoiber

Among these, the role SOCS3 in regulation of IL-6 responses has been studied in detail. IL-6 is a pro-inflammatory cytokine that has been found to play a role in many inflammatory diseases, while IL-10 is a potent anti-inflammatory cytokine, which suppresses gene activation through TLR signalling pathways, but can also inhibit Th1 responses at different levels. While it is known that STAT3 is essential for the biological actions of both IL-6 and IL-10, it was unclear for many years how these two cytokines

Interestingly, an inhibitory role for SOCS3 could only be shown for cytokines binding to the IL-6 receptor whereas the signalling of IL-10 that also stimulates STAT3-activation was unaffected by SOCS3 (Song & Shuai 1998; Lang *et al.* 2003; Yasukawa *et al.* 2003a; Kimura *et al.* 2004; Shuai 2006). This is explained by the ability of SOCS3 to bind to the IL-6 receptor subunit gp130 (Tyr 759) but not to the IL-10 receptor (Nicholson *et al.* 2000; Lehmann *et al.* 2003; Yasukawa *et al.* 2003a). STAT3 activation in response to IL-6 is prolonged in absence of SOCS3. It has been proposed that the sustained activation of STAT3 is essential for the antiinflammatory effect, while transient activation of STAT3 promotes inflammation (Yasukawa

Gp130 is a promiscuous cytokine receptor subunit that mediates signalling by IL-6 and other cytokines such as IL-11, IL-27, leukemia inhibitory factor (LIF) and cardiotrophin-1 (Taga & Kishimoto 1997). Gp130 is present on hematopoietic and nonhematopoietic cells, and its expression can vary depending on the cell's activation status (Andersson *et al.* 1978). Gp130 itself does not bind to cytokines but acts as a co-receptor. When the cytokine (for example IL-6) binds to the IL-6Ra, it triggers a heterodimeric association with gp130 to form a signalling complex (Silver & Hunter 2010). SOCS3 also modulates signalling of gp-130 dependent cytokines. Moreover, the mutation of SOCS3 binding site on the gp130 receptor increased STAT3 activation in response to IL-6 and stimulated IL-10-like anti-inflammatory responses (Ohtani *et al.* 2000; Croker *et al.* 2003; Lang *et al.* 2003; Yasukawa *et al.* 2003a). Such anti-inflammatory responses are abolished when the gp130 Y759 mutated mice possessed a monoallelic deletion of STAT3 (McLoughlin *et al.* 2005; Yoshimura *et al.* 2007). As already mentioned above, SOCS3 inhibits also signalling via non gp130 containing cytokine and hormone receptors such as GCSF, leptin, IL-12 and even IFNs (Song & Shuai 1998; Bousquet

SOCS3 knock out mice die during embryonic life due to placental defects (Marine *et al.* 1999a; Roberts *et al.* 2001). The early death is caused by enhanced LIF signalling, since SOCS3-/- mice showed altered trophoblast differentiation. Animals, in which the SOCS3 deletion in the placenta was rescued by a tetraploid rescue method, showed extended embryonic life. However, rescued SOCS3-/- embryos died due to cardiac hypertrophy

Mice with a conditional knock down of SOCS3 in myeloid cells demonstrated the role of SOCS3 in suppression of IL-6/gp130 signalling. SOCS3-deficient macrophages stimulated with IL-6 displayed an increased magnitude and duration of STAT1 and STAT3 activation

in comparison to controls (Croker *et al.* 2003; Lang *et al.* 2003; Yasukawa *et al.* 2003a).

*et al.* 1999; Bjorbak *et al.* 2000; Shen *et al.* 2000; Hortner *et al.* 2002b).

*et al.* 1999; Dalpke *et al.* 2001).

*et al.* 2003a).

(Takahashi *et al.* 2003).

**4.1 SOCS3 in myeloid cells/granulopoiesis**

could have exactly opposing functions.

Mice deficient for SOCS3 in myeloid cells are resistant to LPS-induced endotoxic shock whereas STAT3 deficient mice are highly sensitive (Takeda *et al.* 1999; Yasukawa *et al.* 2003a). Thus, STAT3 activation mediates anti-inflammatory IL-10 signalling and in the absence of SOCS3, similar anti-inflammatory properties in response to IL-6 (Johnston & O'Shea 2003). Furthermore, macrophages lacking SOCS3 were shown to secrete reduced levels of TNF- and IL-12 after IL-6 and LPS stimulation (Yasukawa *et al.* 2003a).

In a similar manner, DCs deficient for STAT3 contributed to T cell hyper-activation while in the absence of SOCS3, DCs induced a selective expansion of regulatory T cells (Cheng *et al.* 2003; Matsumura *et al.* 2007). An overexpression of SOCS3 in DCs resulted a reduction of expression of co-stimulatory molecules and IL-12. Moreover, SOCS3-deficient and overexpressing DCs had the capacity to suppress EAE development (Li *et al.* 2006; Matsumura *et al.* 2007).

Croker et al. (2003) and Lang et al (2003) on the other hand, using SOCS3 deficient macrophages found an increased STAT1 activation and elevated levels of IFN--regulated genes in response to IL-6.

Fig. 4. SOCS3 inhibits anti-inflammatory and IFN-inducible genes in response to IL-6. Adapted from (Johnston & O'Shea 2003)

Other molecular targets of SOCS3 have been reported: SOCS3 inhibited the activation of TNF-receptor-associated factor 6 (TRAF6) and TGF--activated kinase 1 (TAK1), essential for both TLR– and IL-1-induced responses (Frobose *et al.* 2006). Prele et al. (2006) found no differences in LPS responses of SOCS3 transfected human macrophages. Thus, further studies are required to determine whether there is a direct role for SOCS3 in TLR signalling.

Upon over-expression, SOCS3 was found to bind to the GCSF receptor and reduced STAT3 activation in response to GCSF (Hortner *et al.* 2002b; Hermans *et al.* 2003). In agreement, deletion of SOCS3 in the myeloid or hematopoietic cells increased numbers of neutrophils, which showed increased survival and proliferative capacity (Croker *et al.* 2004; Kimura *et al.* 2004). Following GCSF injection SOCS3-deficient mice developed neutrophilia, and a spectrum of inflammatory pathologies, characterised by neutrophil infiltration in multiples tissues (Croker *et al.* 2008). Overall, SOCS3 regulates survival, growth and activation of neutrophils and an inhibition of SOCS3 may enhance the essential neutrophil recovery after chemotherapy or neutropenia.

"Suppressor of Cytokine Signalling" Molecules in Infection and Inflammation 291

proliferation and suppressive function. The role of SOCS3 on T cell differentiation is still not completely understood but it seems likely that SOCS3 impedes both T reg function (and thereby increasing immune responses) and Th17 differentiation (impairing inflammatory

SOCS3 has also been shown to modulate B cell development by affecting the lodging of precursor B cells to the bone marrow, via the regulation of responses to CXCL12 (Le *et al.*

Infections are accompanied by tissue insulin resistance, as manifested by worsening of metabolic control in diabetic patients and decreased glucose tolerance in non-diabetic subjects. A propensity towards glucose intolerance and diabetes has been documented in patients with chronic HCV infection. Diabetes patients are more susceptible to infections such as tuberculosis or candidiasis. These associated diseases could be linked to SOCS expressin. As stated below pathogen molecules can stimulate SOCS3 expression. SOCS3 binds to the leptin and insulin receptor and inhibits STAT3 activation in response to leptin and insulin respectively (Clement *et al.* 1998; Bjorbak *et al.* 2000; Emanuelli *et al.* 2000; Eyckerman *et al.* 2000; Chen *et al.* 2006). Leptin is an adipocyte-derived hormone that regulates food intake and energy homeostasis (Flier 2004). Leptin-resistance, common in human obesity and in acquired obesity in rodents, may be regulated by SOCS3 (Bjorbaek *et al.* 1998). High fat diet fed SOCS3+/- mice as well as neural cell-specific SOCS3- knockdown mice showed reduced diet-induced obesity, together with increased leptin and insulin sensitivity (Howard *et al.* 2004; Mori *et al.* 2004; Kievit *et al.* 2006). Furthermore, SOCS3 deficient adipocytes were protected from TNF--induced insulin resistance (Shi *et al.* 2004). However, mice with a knock down of SOCS3 in hepatocytes displayed increased insulin sensitivity in the liver but developed obesity and systemic insulin resistance with age (Torisu *et al.* 2007). This insulin resistance was accounted by increased inflammatory responses due to the lack of SOCS3. Accordingly, overexpression of SOCS3 in the liver of mice induced insulin resistance, whereas silencing of SOCS3 with antisense oligonucleotides in obese diabetic mice improved insulin sensitivity (Ueki *et al.* 2004a; Ueki *et al.* 2004b). The consistency of the effect of SOCS3 in different approaches on both leptin and insulin resistance indicates SOCS3 as a unique mediator of both obesity and

Similar to SOCS1, SOCS3 expression can be stimulated by both cytokines but also TLR agonists. Additionally, several pathogens including viruses, bacteria and parasites have been shown to stimulate SOCS3 expression. But due to multiple binding partners of SOCS3,

SOCS3 was found to inhibit type I IFN signalling although SOCS3 was less effective than SOCS1 (Song & Shuai 1998; Shen *et al.* 2000). Infection with several viruses induced SOCS3 expression that correlated with reduced STAT1 activation in response to IFN-. HSV stimulated SOCS3 in different human cell lines but not in the B-cell line AKATA. In contrast to SOCS3-expressing cell lines, infected AKATA cells showed no impaired IFN-

responses, fig. 5).

insulin resistance.

**4.2.1 Viral infections**

**4.1.2 SOCS3 in leptin and insulin resistance**

**4.2 Role of SOCS3 in infectious diseases and inflammation**

it is hard to predict the role of SOCS3 in different infections.

2007).

#### **4.1.1 SOCS3 in T cells**

T cell development in the thymus is unaffected by SOCS3 (Chen *et al.* 2006). However, SOCS3 might play an active role in T cell proliferation and differentiation.

The proliferation of T cells was found to be regulated by SOCS3. T cells overexpressing SOCS3 showed reduced proliferation to mitogens and anti-CD28 (Banerjee *et al.* 2002; Matsumoto *et al.* 2003). On the other hand, SOCS3-deficient CD8 T cells also showed enhanced anti-CD3-induced proliferation. Neutralization of IL-27 also limited T cell proliferation indicating that IL-27 responses are impaired by SOCS3 and account for diminished T cell proliferation (Brender *et al.* 2007).

There is a reciprocal relation between SOCS1 and SOCS3 expression levels in T cells, with high SOCS1 and low SOCS3 expression in Th1 cells whereas high SOCS3 and low SOCS1 expression was found in Th2 cells (Egwuagu *et al.* 2002; Seki *et al.* 2003). However, there are conflicting results whether levels of SOCS3 actively influences the Th1/Th2 balance. Overexpression of SOCS3 in murine T cells resulted in elevated Th2 and decreased Th1 responses during allergy. Furthermore, a reduced Th2 response characterized by decreased IL-4 and increased IFN- production was shown in SOCS3+/- mice (Seki *et al.* 2003). A SOCS3 mediated regulation of STAT5 activation was reported to account for the altered Th1/Th2 balance. Another explanation for reduced Th2 responses was suggested by Kinjyo et al. (2006) using Lck SOCS3fl/fl mice, in which Th3-like T cells with higher IL-10 and TGF levels were found. These cytokines may then suppress Th1 responses accounting for the decreased Th1/Th2 ratio in SOCS3 deficient mice. However this observation was not confirmed by others: mice with a specific deletion of SOCS3 in the lymphoid cells were reported to have an unaltered Th1 and Th2 cell differentiation (Chen *et al.* 2006). Instead, loss of SOCS3 resulted in enhanced Th17 generation and enhanced STAT3 activation in response to IL-23. Th17 is a T cell subset that produces IL-17 and plays a key role in different autoimmune diseases and in the defense against fungal infections. STAT3 interacted with the *IL17A* and *IL-17F* promoter (Kinjyo *et al.* 2006). TGF--mediated the inhibition of SOCS3 expression and therefore prolonged STAT3 activation promoting Th17 development (Qin *et al.* 2009).

The importance of SOCS3 in impairing Th17 development was further confirmed in a study of an atherosclerosis model in mice with a T cell specific SOCS3 knock down and in a rheumatoid arthritis model in mice lacking SOCS3 in hematopoietic and endothelial cells (Wong *et al.* 2006; Taleb *et al.* 2009).

Fig. 5. SOCS3 hampers Th17 cell development

Regulatory T cells, a subset of CD4 T cells that suppress T cell-mediated immune responses and maintain tolerance to self-antigens, were found to be deficient for SOCS3 protein expression (Pillemer *et al.* 2007). Over-expression of SOCS3 in T regs impaired their proliferation and suppressive function. The role of SOCS3 on T cell differentiation is still not completely understood but it seems likely that SOCS3 impedes both T reg function (and thereby increasing immune responses) and Th17 differentiation (impairing inflammatory responses, fig. 5).

SOCS3 has also been shown to modulate B cell development by affecting the lodging of precursor B cells to the bone marrow, via the regulation of responses to CXCL12 (Le *et al.* 2007).

#### **4.1.2 SOCS3 in leptin and insulin resistance**

Inflammation, Chronic Diseases and Cancer –

290 Cell and Molecular Biology, Immunology and Clinical Bases

T cell development in the thymus is unaffected by SOCS3 (Chen *et al.* 2006). However,

The proliferation of T cells was found to be regulated by SOCS3. T cells overexpressing SOCS3 showed reduced proliferation to mitogens and anti-CD28 (Banerjee *et al.* 2002; Matsumoto *et al.* 2003). On the other hand, SOCS3-deficient CD8 T cells also showed enhanced anti-CD3-induced proliferation. Neutralization of IL-27 also limited T cell proliferation indicating that IL-27 responses are impaired by SOCS3 and account for

There is a reciprocal relation between SOCS1 and SOCS3 expression levels in T cells, with high SOCS1 and low SOCS3 expression in Th1 cells whereas high SOCS3 and low SOCS1 expression was found in Th2 cells (Egwuagu *et al.* 2002; Seki *et al.* 2003). However, there are conflicting results whether levels of SOCS3 actively influences the Th1/Th2 balance. Overexpression of SOCS3 in murine T cells resulted in elevated Th2 and decreased Th1 responses during allergy. Furthermore, a reduced Th2 response characterized by decreased IL-4 and increased IFN- production was shown in SOCS3+/- mice (Seki *et al.* 2003). A SOCS3 mediated regulation of STAT5 activation was reported to account for the altered Th1/Th2 balance. Another explanation for reduced Th2 responses was suggested by Kinjyo et al. (2006) using Lck SOCS3fl/fl mice, in which Th3-like T cells with higher IL-10 and TGF levels were found. These cytokines may then suppress Th1 responses accounting for the decreased Th1/Th2 ratio in SOCS3 deficient mice. However this observation was not confirmed by others: mice with a specific deletion of SOCS3 in the lymphoid cells were reported to have an unaltered Th1 and Th2 cell differentiation (Chen *et al.* 2006). Instead, loss of SOCS3 resulted in enhanced Th17 generation and enhanced STAT3 activation in response to IL-23. Th17 is a T cell subset that produces IL-17 and plays a key role in different autoimmune diseases and in the defense against fungal infections. STAT3 interacted with the *IL17A* and *IL-17F* promoter (Kinjyo *et al.* 2006). TGF--mediated the inhibition of SOCS3 expression and therefore prolonged STAT3 activation promoting Th17

The importance of SOCS3 in impairing Th17 development was further confirmed in a study of an atherosclerosis model in mice with a T cell specific SOCS3 knock down and in a rheumatoid arthritis model in mice lacking SOCS3 in hematopoietic and endothelial cells

Regulatory T cells, a subset of CD4 T cells that suppress T cell-mediated immune responses and maintain tolerance to self-antigens, were found to be deficient for SOCS3 protein expression (Pillemer *et al.* 2007). Over-expression of SOCS3 in T regs impaired their

SOCS3 might play an active role in T cell proliferation and differentiation.

diminished T cell proliferation (Brender *et al.* 2007).

**4.1.1 SOCS3 in T cells**

development (Qin *et al.* 2009).

(Wong *et al.* 2006; Taleb *et al.* 2009).

Fig. 5. SOCS3 hampers Th17 cell development

Infections are accompanied by tissue insulin resistance, as manifested by worsening of metabolic control in diabetic patients and decreased glucose tolerance in non-diabetic subjects. A propensity towards glucose intolerance and diabetes has been documented in patients with chronic HCV infection. Diabetes patients are more susceptible to infections such as tuberculosis or candidiasis. These associated diseases could be linked to SOCS expressin. As stated below pathogen molecules can stimulate SOCS3 expression. SOCS3 binds to the leptin and insulin receptor and inhibits STAT3 activation in response to leptin and insulin respectively (Clement *et al.* 1998; Bjorbak *et al.* 2000; Emanuelli *et al.* 2000; Eyckerman *et al.* 2000; Chen *et al.* 2006). Leptin is an adipocyte-derived hormone that regulates food intake and energy homeostasis (Flier 2004). Leptin-resistance, common in human obesity and in acquired obesity in rodents, may be regulated by SOCS3 (Bjorbaek *et al.* 1998). High fat diet fed SOCS3+/- mice as well as neural cell-specific SOCS3- knockdown mice showed reduced diet-induced obesity, together with increased leptin and insulin sensitivity (Howard *et al.* 2004; Mori *et al.* 2004; Kievit *et al.* 2006). Furthermore, SOCS3 deficient adipocytes were protected from TNF--induced insulin resistance (Shi *et al.* 2004). However, mice with a knock down of SOCS3 in hepatocytes displayed increased insulin sensitivity in the liver but developed obesity and systemic insulin resistance with age (Torisu *et al.* 2007). This insulin resistance was accounted by increased inflammatory responses due to the lack of SOCS3. Accordingly, overexpression of SOCS3 in the liver of mice induced insulin resistance, whereas silencing of SOCS3 with antisense oligonucleotides in obese diabetic mice improved insulin sensitivity (Ueki *et al.* 2004a; Ueki *et al.* 2004b). The consistency of the effect of SOCS3 in different approaches on both leptin and insulin resistance indicates SOCS3 as a unique mediator of both obesity and insulin resistance.

#### **4.2 Role of SOCS3 in infectious diseases and inflammation**

Similar to SOCS1, SOCS3 expression can be stimulated by both cytokines but also TLR agonists. Additionally, several pathogens including viruses, bacteria and parasites have been shown to stimulate SOCS3 expression. But due to multiple binding partners of SOCS3, it is hard to predict the role of SOCS3 in different infections.

#### **4.2.1 Viral infections**

SOCS3 was found to inhibit type I IFN signalling although SOCS3 was less effective than SOCS1 (Song & Shuai 1998; Shen *et al.* 2000). Infection with several viruses induced SOCS3 expression that correlated with reduced STAT1 activation in response to IFN-. HSV stimulated SOCS3 in different human cell lines but not in the B-cell line AKATA. In contrast to SOCS3-expressing cell lines, infected AKATA cells showed no impaired IFN-

"Suppressor of Cytokine Signalling" Molecules in Infection and Inflammation 293

SFV IFN- signalling worsened HTLV-1 IFN- signalling. worsened VSV IFN- signalling. worsened HSV-1 IFN- signalling. worsened Vaccinia Virus immune responses improved improved

HIV-1 Ubiquitination of HIVgag worsened *C. pneumoniae* IFN- signalling improved worsened *M. tuberculosis* – *M. bovis* BCG IL-12 signalling improved worsened *P. berghi* ANKA ??? worsened worsened *L. major*  IFN- signalling improved worsened *T. gondii*  IFN- signalling worsened *B. malayi* Th1/Th2 ratio worsened

HSV-1 IFN- signalling. worsened

Influenza A Virus IFN- signalling worsened LCMV T cell activation worsened worsened

by T cells

SOCS expression is tightly regulated to prevent inflammation while maintaining protective anti-microbial responses. Pathogens are able to potently stimulate SOCS1 and 3 protein expression following infection and hijack SOCS function promoting their survival. In most infections studied, the stimulation of SOCS1 and SOCS3 expression by infectious agents resulted in a worsened pathogen control even though the nature of immune inhibition by the SOCS proteins differed. Thus, SOCS proteins can be manipulated to increase innate immune mechanisms, but also the triggering and the magnitude of adaptive immune responses. This converts SOCS proteins into very attractive therapeutic targets. However manipulation of SOCS levels always bears the risk of increased inflammatory responses. A better understanding of the role of SOCS in infections is required to better gauge SOCS

Adams, T.E., Hansen, J.A., Starr, R., Nicola, N.A., Hilton, D.J. & Billestrup, N. (1998) Growth

hormone preferentially induces the rapid, transient expression of SOCS-3, a novel inhibitor of cytokine receptor signaling. *The Journal of Biological Chemistry* 273, 1285-

dendritic cells

RSV IFN- signalling. HIV-1 IFN- signalling

*L. major* TGF- IL-10 production

*T. gondii (gp130 mutation)* IL-12 induction in

Table 1. Role of SOCS molecules during microbial infections.

manipulation leading to potentially new therapies.

**pathogen mechanism pathology pathogen**

**control**

? improved

improved improved

**S O C S 1**

**S O C S 3**

**5. Conclusion**

**6. References**

1287.

mediated STAT1 activation during HSV infection. Silencing of SOCS3 using anti-sense nucleotides significantly hampered replication of HSV (Yokota *et al.* 2005). Similar observations were done during Influenza A, HCV and Epstein Barr virus infections indicating that SOCS3 expression facilitates viral infections (Bode *et al.* 2003; Le Goffic *et al.* 2007; Pothlichet *et al.* 2008; Michaud *et al.* 2010).

Interestingly, mice with a T cell-specific SOCS3 deletion showed increased T cell activation and viral clearance without development of immunopathology during the infection with lymphocytic chriomeningitis virus (LCMV) (Pellegrini *et al.* 2011). Treatment of LCMVinfected mice with IL-7 repressed SOCS3 expression and promoted IL-6 production resulting in enhanced T cell effector functions, numbers and viral clearance. However, the deletion of SOCS3 in all hematopoietic cells induced neutrophilia and early lethality in LCMV-infected mice (Pellegrini *et al.* 2011).

Patients with genotype 1 HCV infection tend to have higher levels of SOCS3 expression, providing a rationale for their propensity toward a lack of therapeutic response (Kim *et al.* 2009). Moreover, one SOCS3 genotype (–4874 AA) expressed SOCS3 at elevated levels and showed a poorerresponse to therapy (Persico *et al.* 2008).

#### **4.2.2 Bacterial infections**

The infection of macrophages with *Salmonella enterica* has been shown to increment SOCS3 but not SOCS1 expression (Uchiya & Nikai 2005). SOCS3 stimulation was dependent on the *Salmonella* pathogenicity island 2 important for bacterial virulence. Infected macrophages showed impaired STAT1 and STAT3 activation in response to IL-6 or IFN- respectively, which might prevent effective bacterial killing.

Infection of macrophages with *Mycobacterium avium* or *M. bovis* raised SOCS1 and SOCS3 levels in a TLR2-NOTCH1 dependent pathway (Imai *et al.* 2003; Vazquez *et al.* 2006; Narayana & Balaji 2008).

Patients with active tuberculosis were found to have higher SOCS3 expression levels in whole blood and T cells in comparison to latently infected controls or to patients after chemotherapy (Mistry *et al.* 2007; Jacobsen *et al.* 2010). We observed that SOCS3 expression in blood non-T cells from patients with TB was negatively associated with severity of disease (Masood *et al*, 2011). Atogether the role of SOCS3 in bacterial infections is far from clear.

#### **4.2.3 Parasites**

Little is known about the role of SOCS3 in parasites. Infection of macrophages with the parasite *Leishmania dovani* was shown to stimulate SOCS3 expression. SOCS3 levels associated with reduced STAT1 activation after IFN- stimulation and therefore with reduced protection (Bertholet *et al.* 2003).

On the contrary, mice lacking SOCS3 in T cells were more susceptible to *Leishmania major* infections. SOCS3-deficiency in T cells led to increased anti-inflammatory TGF- and IL-10 secretion by T cells and reduced immunoglobulin levels (Kinjyo *et al.* 2006).

Mice bearing a mutation in the SOCS3 binding site of the gp130 receptor displayed increased susceptibility to *Toxoplasma gondii*. A decreased IL-12 production by DCs resulted in reduced IFN- secretion by NK cells of these mice. Addition of IL-12 as well as neutralization of IL-6 could restore the wild type phenotype indicating that an altered IL-6 signalling was responsible for increased susceptibility (Silver *et al.* 2011).

Table 1. Role of SOCS molecules during microbial infections.

#### **5. Conclusion**

Inflammation, Chronic Diseases and Cancer –

292 Cell and Molecular Biology, Immunology and Clinical Bases

mediated STAT1 activation during HSV infection. Silencing of SOCS3 using anti-sense nucleotides significantly hampered replication of HSV (Yokota *et al.* 2005). Similar observations were done during Influenza A, HCV and Epstein Barr virus infections indicating that SOCS3 expression facilitates viral infections (Bode *et al.* 2003; Le Goffic *et al.*

Interestingly, mice with a T cell-specific SOCS3 deletion showed increased T cell activation and viral clearance without development of immunopathology during the infection with lymphocytic chriomeningitis virus (LCMV) (Pellegrini *et al.* 2011). Treatment of LCMVinfected mice with IL-7 repressed SOCS3 expression and promoted IL-6 production resulting in enhanced T cell effector functions, numbers and viral clearance. However, the deletion of SOCS3 in all hematopoietic cells induced neutrophilia and early lethality in

Patients with genotype 1 HCV infection tend to have higher levels of SOCS3 expression, providing a rationale for their propensity toward a lack of therapeutic response (Kim *et al.* 2009). Moreover, one SOCS3 genotype (–4874 AA) expressed SOCS3 at elevated levels and

The infection of macrophages with *Salmonella enterica* has been shown to increment SOCS3 but not SOCS1 expression (Uchiya & Nikai 2005). SOCS3 stimulation was dependent on the *Salmonella* pathogenicity island 2 important for bacterial virulence. Infected macrophages showed impaired STAT1 and STAT3 activation in response to IL-6 or IFN- respectively,

Infection of macrophages with *Mycobacterium avium* or *M. bovis* raised SOCS1 and SOCS3 levels in a TLR2-NOTCH1 dependent pathway (Imai *et al.* 2003; Vazquez *et al.* 2006;

Patients with active tuberculosis were found to have higher SOCS3 expression levels in whole blood and T cells in comparison to latently infected controls or to patients after chemotherapy (Mistry *et al.* 2007; Jacobsen *et al.* 2010). We observed that SOCS3 expression in blood non-T cells from patients with TB was negatively associated with severity of disease (Masood *et al*, 2011). Atogether the role of SOCS3 in bacterial infections is far from

Little is known about the role of SOCS3 in parasites. Infection of macrophages with the parasite *Leishmania dovani* was shown to stimulate SOCS3 expression. SOCS3 levels associated with reduced STAT1 activation after IFN- stimulation and therefore with

On the contrary, mice lacking SOCS3 in T cells were more susceptible to *Leishmania major* infections. SOCS3-deficiency in T cells led to increased anti-inflammatory TGF- and IL-10

Mice bearing a mutation in the SOCS3 binding site of the gp130 receptor displayed increased susceptibility to *Toxoplasma gondii*. A decreased IL-12 production by DCs resulted in reduced IFN- secretion by NK cells of these mice. Addition of IL-12 as well as neutralization of IL-6 could restore the wild type phenotype indicating that an altered IL-6

secretion by T cells and reduced immunoglobulin levels (Kinjyo *et al.* 2006).

signalling was responsible for increased susceptibility (Silver *et al.* 2011).

2007; Pothlichet *et al.* 2008; Michaud *et al.* 2010).

LCMV-infected mice (Pellegrini *et al.* 2011).

which might prevent effective bacterial killing.

reduced protection (Bertholet *et al.* 2003).

**4.2.2 Bacterial infections**

Narayana & Balaji 2008).

clear.

**4.2.3 Parasites**

showed a poorerresponse to therapy (Persico *et al.* 2008).

SOCS expression is tightly regulated to prevent inflammation while maintaining protective anti-microbial responses. Pathogens are able to potently stimulate SOCS1 and 3 protein expression following infection and hijack SOCS function promoting their survival. In most infections studied, the stimulation of SOCS1 and SOCS3 expression by infectious agents resulted in a worsened pathogen control even though the nature of immune inhibition by the SOCS proteins differed. Thus, SOCS proteins can be manipulated to increase innate immune mechanisms, but also the triggering and the magnitude of adaptive immune responses. This converts SOCS proteins into very attractive therapeutic targets. However manipulation of SOCS levels always bears the risk of increased inflammatory responses. A better understanding of the role of SOCS in infections is required to better gauge SOCS manipulation leading to potentially new therapies.

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**14**

**Role of the Neutrophil NADPH Oxidase and**

Reactive oxygen species (ROS) are low-molecular-weight inorganic and highly reactive compounds that are produced during normal aerobic cell metabolism. At low level, ROS have a transient mode of action and important physiological roles in maintaining intracellular cell redox status, physical-chemical properties of membranes, protein kinase and transcriptional factor activities. They participate in the regulation of many aspects of fundamental cellular function, including growth-specific and migration-related signalling pathways, gene expression, GTPase-dependent cytoskeletal rearrangements, cell proliferation and apoptosis. Multiple endogenous macromolecules, participating in cellular signalling networks, bear redox-active moieties (e.g., methionine, cysteine, guanine) at functional regions that render them sensitive to ROS. Consequently, ROS emerge, not only as second messengers, but also as diffusible modulators able to produce stable secondary

A major source of ROS are phagocytic cells such as polymorphonuclear neutrophils that are found activated and adherent to the endothelium or migrating through the extravascular tissue matrix. ROS produced by neutrophils are designed to kill the invading pathogens and have an important role in priming the immune system. The phagocyte NADPH oxidase, also referred as NOX2, is composed of several membane-bound and cytosolic subunits that assemble rapidly in the phagosomal or plasma membrane (Fig. 1). This allows the concentrated release of ROS at sites of inflammation where the pathogen is located (Ohno et al., 1982) and diffusion over short distances, enabling these second messengers to act on adjacent neighbouring targets. Since many years, it is known that ROS interactions with lipids contribute to disturbances in cell structure (Thaw et al., 1983) and alterations of functional activities (Bellomo et al., 1982; Poot et al., 1988) enhancing the ability of the

Under physiological conditions, ROS are eliminated by several defence mechanisms (e.g., antioxidant enzymes) to maintain a normal intracellular redox environment and control the

However, when these protective mechanisms are overwhelmed by excessive amounts of ROS, due to inappropriate activation of phagocytes or alterations of the antioxidant defence system, a situation of oxidative stress can occur resulting in abnormal physiological

phagocytes to kill invading microorganisms (Quinn et al., 1995).

**1. Introduction**

signalling molecules.

signalling cascades.

**S100A8/A9 in the Pathophysiology**

Sabrina Bréchard, Véronique Schenten and Eric J. Tschirhart

 **of Chronic Inflammation**

*University of Luxembourg*

*Luxembourg*

SOCS box of suppressor of cytokine signaling-1 is important for inhibition of cytokine action in vivo. *Proceedings of the National Academy of Sciences of the United States of America* 98, 13261-13265.

### **Role of the Neutrophil NADPH Oxidase and S100A8/A9 in the Pathophysiology of Chronic Inflammation**

Sabrina Bréchard, Véronique Schenten and Eric J. Tschirhart *University of Luxembourg Luxembourg*

#### **1. Introduction**

Inflammation, Chronic Diseases and Cancer –

306 Cell and Molecular Biology, Immunology and Clinical Bases

Zhang, Y., Ma, C.J., Ni, L., Zhang, C.L., Wu, X.Y., Kumaraguru, U., Li, C.F., Moorman, J.P. &

Zimmermann, S., Murray, P.J., Heeg, K. & Dalpke, A.H. (2006) Induction of suppressor of

Zitzmann, K., Brand, S., De Toni, E.N., Baehs, S., Goke, B., Meinecke, J., Spottl, G., Meyer,

in hepatitis C virus infection. *Journal of Immunology* 186, 3093-3103.

*States of America* 98, 13261-13265.

1847.

67, 5025-5032.

SOCS box of suppressor of cytokine signaling-1 is important for inhibition of cytokine action in vivo. *Proceedings of the National Academy of Sciences of the United*

Yao, Z.Q. (2011) Cross-talk between programmed death-1 and suppressor of cytokine signaling-1 in inhibition of IL-12 production by monocytes/macrophages

cytokine signaling-1 by Toxoplasma gondii contributes to immune evasion in macrophages by blocking IFN-gamma signaling. *Journal of Immunology* 176, 1840-

H.H. & Auernhammer, C.J. (2007) SOCS1 silencing enhances antitumor activity of type I IFNs by regulating apoptosis in neuroendocrine tumor cells. *Cancer Research*

> Reactive oxygen species (ROS) are low-molecular-weight inorganic and highly reactive compounds that are produced during normal aerobic cell metabolism. At low level, ROS have a transient mode of action and important physiological roles in maintaining intracellular cell redox status, physical-chemical properties of membranes, protein kinase and transcriptional factor activities. They participate in the regulation of many aspects of fundamental cellular function, including growth-specific and migration-related signalling pathways, gene expression, GTPase-dependent cytoskeletal rearrangements, cell proliferation and apoptosis. Multiple endogenous macromolecules, participating in cellular signalling networks, bear redox-active moieties (e.g., methionine, cysteine, guanine) at functional regions that render them sensitive to ROS. Consequently, ROS emerge, not only as second messengers, but also as diffusible modulators able to produce stable secondary signalling molecules.

> A major source of ROS are phagocytic cells such as polymorphonuclear neutrophils that are found activated and adherent to the endothelium or migrating through the extravascular tissue matrix. ROS produced by neutrophils are designed to kill the invading pathogens and have an important role in priming the immune system. The phagocyte NADPH oxidase, also referred as NOX2, is composed of several membane-bound and cytosolic subunits that assemble rapidly in the phagosomal or plasma membrane (Fig. 1). This allows the concentrated release of ROS at sites of inflammation where the pathogen is located (Ohno et al., 1982) and diffusion over short distances, enabling these second messengers to act on adjacent neighbouring targets. Since many years, it is known that ROS interactions with lipids contribute to disturbances in cell structure (Thaw et al., 1983) and alterations of functional activities (Bellomo et al., 1982; Poot et al., 1988) enhancing the ability of the phagocytes to kill invading microorganisms (Quinn et al., 1995).

> Under physiological conditions, ROS are eliminated by several defence mechanisms (e.g., antioxidant enzymes) to maintain a normal intracellular redox environment and control the signalling cascades.

> However, when these protective mechanisms are overwhelmed by excessive amounts of ROS, due to inappropriate activation of phagocytes or alterations of the antioxidant defence system, a situation of oxidative stress can occur resulting in abnormal physiological

Role of the Neutrophil NADPH Oxidase and

mutagenic in mammalian cells.

(Jessup et al., 1986; Vindis et al., 2007).

**2.1.2 Protein oxidation**

Schewe, 2000).

S100A8/A9 in the Pathophysiology of Chronic Inflammation 309

membrane modifying its physical properties and causing changes in fluidity and permeability, inhibition of metabolic processes, and alterations of ion transport (Nigam &

Polyunsaturated fatty acid residues of phospholipids nearby membrane were found to be extremely sensitive to oxidation by the highly reactive hydroxyl radical (Siems et al., 1995). The initial reaction of hydroxyl radical with polyunsaturated fatty acids produces an alkyl radical, which in turn reacts with molecular oxygen to form a peroxyl radical in a perpetuating chain reaction. Once formed, peroxyl radicals can undergo subsequent cyclization to generate endoperoxides, which leads to the final production of malondialdehyde (Mao et al., 1999; Marnett, 2002). Malondialdehyde reacts with DNA, predominantly with deoxyguanosine, to form exocyclic adducts most of which are

The major aldehyde product of lipid peroxidation other than malondialdehyde is 4 hydroxy-2-nonenal (4-HNE) that is a weakly mutagenic metabolite but with a high cytotoxicity. The cytotoxic effects are thought to be due to the ability of 4-HNE to react readily with sulfhydryl groups by a Michael addition mechanism to form stable thioether derivatives (Esterbauer et al., 1975; Esterbauer et al., 1991). 4-HNE is a potent inhibitor of sulfhydryl enzymes, such as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) via modification of the cysteine residue (Cys-149) at the catalytic site (Uchida & Stadtman, 1993). Later, it was suggested that 4-HNE inactivation of GAPDH is not due to the modification of the catalytic center but to the sequential and selective modification of amino acids (His-164, Cys-281, Cys-244, His-327 and Lys-331) primarily

4-HNE reacts also with selective histidine residues of proteins including insulin (which lacks cysteine) contributing to the cytotoxic action of 4-HNE (Uchida & Stadtman, 1992). In certain proteins, lysine c-amino groups, but not histidine, is the major target amino acid of 4-HNE. Lysine modification in glucose-6-phosphate dehydrogenase via Schiff-base formation is associated with a loss of enzyme activity (Sweda et al., 1993) and 4-HNE modification on low density lipoproteins have been implicated in the pathophysiology of atherosclerosis

Many different types of oxidative modification of proteins can be induced by ROS. Cysteine and methionine residues of proteins are highly susceptible to oxidation by various forms of ROS. Oxidation of cysteine residues can lead to the formation of a mixed disulphide between protein thiol groups and low molecular weight thiol compounds (reversible S-glutathiolation). Carbonyl groups are major products of ROS-mediated oxidation proteins and the concentration of these derivatives is an adapted marker of protein oxidation (Berlett et al., 1997; Chevion et al., 2000). Protein modifications elicited by oxidative attack on lysine, arginine, proline or threonine, or by secondary reaction of cysteine, histidine or lysine residues with carbonyl compounds can result in the formation of protein derivatives possessing highly reactive carbonyl groups such as aldehydes and ketones (Berlett et al., 1997). Oxidation of some critical methionine residues causes a complete inhibition of actin polymerization and

Oxidative modification of proteins by ROS is implicated in the etiology and/or progression of a broad variety of age-related degenerative pathologies, atherosclerosis, muscular dystrophy, rheumatoid arthritis (Chevion et al., 2000; Moreira et al., 2010; Stadtman &

destabilization of the structure of actin filaments (Dalle-Donne et al., 2002).

located in the surface of the GAPDH molecule (Ishii et al., 2003).

responses. During a prolonged period, focal regions of the endothelium can be exposed to aberrant levels of ROS, which in turn cause the destruction of healthy tissue. Indeed, at high concentrations and continued exposure, ROS can damage all types of biomolecules including DNA, lipids, carbohydrates and proteins.

Fig. 1. Assembly and activation of the phagocyte NADPH oxidase, NOX2 (for reviews, see Babior et al., 2002; Sheppard et al., 2005). NOX2 activation is mediated by the assembly of cytosolic subunits (p40phox, p47phox, and p67phox) with the membrane subunits (p22phox and gp91phox constituting the flavocytochrome b558). Phosphorylation of p47phox, by protein kinases, is notably required for translocation of p47phox to the membrane and binding to p22phox. The guanine nucleotide exchange factor (GEF) triggers GTP binding to Rac promoting the membrane translocation and the binding to p67phox necessary to the assembly of active NOX2 and superoxide anion production.

#### **2. Toxicity of neutrophil NADPH oxidase (NOX2)-mediated release of ROS: Pathophysiological roles**

#### **2.1 Molecular damage caused by ROS**

Irreversible oxidative modification of macromolecules including proteins, lipids, DNA and carbohydrates are typically viewed as the primary cellular targets of ROS and contribute to cell injury. Consequently, oxidized biomolecules are linked to the pathophysiology of multiple chronic human diseases and are the most commonly used biomarkers of oxidative damage isolated from tissues and biological fluids (for review, see Dalle-Donne et al., 2006).

#### **2.1.1 Lipid peroxidation**

Lipid peroxidation is a complex process in which oxidants, generated by NADPH oxidase of activated neutrophils, react the most abundantly with polyunsaturated fatty acids (e.g., linoleic acid and arachidonic acid) to form a variety of products including aldehydes, lipid radicals and hydrocarbons. Peroxidation of lipids can disrupt the organization of the

308 Cell and Molecular Biology, Immunology and Clinical Bases

responses. During a prolonged period, focal regions of the endothelium can be exposed to aberrant levels of ROS, which in turn cause the destruction of healthy tissue. Indeed, at high concentrations and continued exposure, ROS can damage all types of biomolecules

p22phox

Fig. 1. Assembly and activation of the phagocyte NADPH oxidase, NOX2 (for reviews, see Babior et al., 2002; Sheppard et al., 2005). NOX2 activation is mediated by the assembly of cytosolic subunits (p40phox, p47phox, and p67phox) with the membrane subunits (p22phox and gp91phox constituting the flavocytochrome b558). Phosphorylation of p47phox, by protein kinases, is notably required for translocation of p47phox to the membrane and binding to p22phox. The guanine nucleotide exchange factor (GEF) triggers GTP binding to Rac

promoting the membrane translocation and the binding to p67phox necessary to the assembly

**2. Toxicity of neutrophil NADPH oxidase (NOX2)-mediated release of ROS:**

Irreversible oxidative modification of macromolecules including proteins, lipids, DNA and carbohydrates are typically viewed as the primary cellular targets of ROS and contribute to cell injury. Consequently, oxidized biomolecules are linked to the pathophysiology of multiple chronic human diseases and are the most commonly used biomarkers of oxidative damage isolated from tissues and biological fluids (for review, see Dalle-Donne et al., 2006).

Lipid peroxidation is a complex process in which oxidants, generated by NADPH oxidase of activated neutrophils, react the most abundantly with polyunsaturated fatty acids (e.g., linoleic acid and arachidonic acid) to form a variety of products including aldehydes, lipid radicals and hydrocarbons. Peroxidation of lipids can disrupt the organization of the

O2 O2

gp91phox

.-

Rac-GDP

Rac-GTP

Rac-GTP

> Rho-GDI

**GEF**

including DNA, lipids, carbohydrates and proteins.

of active NOX2 and superoxide anion production.

**Pathophysiological roles**

**2.1.1 Lipid peroxidation**

**2.1 Molecular damage caused by ROS**

membrane modifying its physical properties and causing changes in fluidity and permeability, inhibition of metabolic processes, and alterations of ion transport (Nigam & Schewe, 2000).

Polyunsaturated fatty acid residues of phospholipids nearby membrane were found to be extremely sensitive to oxidation by the highly reactive hydroxyl radical (Siems et al., 1995). The initial reaction of hydroxyl radical with polyunsaturated fatty acids produces an alkyl radical, which in turn reacts with molecular oxygen to form a peroxyl radical in a perpetuating chain reaction. Once formed, peroxyl radicals can undergo subsequent cyclization to generate endoperoxides, which leads to the final production of malondialdehyde (Mao et al., 1999; Marnett, 2002). Malondialdehyde reacts with DNA, predominantly with deoxyguanosine, to form exocyclic adducts most of which are mutagenic in mammalian cells.

The major aldehyde product of lipid peroxidation other than malondialdehyde is 4 hydroxy-2-nonenal (4-HNE) that is a weakly mutagenic metabolite but with a high cytotoxicity. The cytotoxic effects are thought to be due to the ability of 4-HNE to react readily with sulfhydryl groups by a Michael addition mechanism to form stable thioether derivatives (Esterbauer et al., 1975; Esterbauer et al., 1991). 4-HNE is a potent inhibitor of sulfhydryl enzymes, such as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) via modification of the cysteine residue (Cys-149) at the catalytic site (Uchida & Stadtman, 1993). Later, it was suggested that 4-HNE inactivation of GAPDH is not due to the modification of the catalytic center but to the sequential and selective modification of amino acids (His-164, Cys-281, Cys-244, His-327 and Lys-331) primarily located in the surface of the GAPDH molecule (Ishii et al., 2003).

4-HNE reacts also with selective histidine residues of proteins including insulin (which lacks cysteine) contributing to the cytotoxic action of 4-HNE (Uchida & Stadtman, 1992). In certain proteins, lysine c-amino groups, but not histidine, is the major target amino acid of 4-HNE. Lysine modification in glucose-6-phosphate dehydrogenase via Schiff-base formation is associated with a loss of enzyme activity (Sweda et al., 1993) and 4-HNE modification on low density lipoproteins have been implicated in the pathophysiology of atherosclerosis (Jessup et al., 1986; Vindis et al., 2007).

#### **2.1.2 Protein oxidation**

Many different types of oxidative modification of proteins can be induced by ROS. Cysteine and methionine residues of proteins are highly susceptible to oxidation by various forms of ROS. Oxidation of cysteine residues can lead to the formation of a mixed disulphide between protein thiol groups and low molecular weight thiol compounds (reversible S-glutathiolation). Carbonyl groups are major products of ROS-mediated oxidation proteins and the concentration of these derivatives is an adapted marker of protein oxidation (Berlett et al., 1997; Chevion et al., 2000). Protein modifications elicited by oxidative attack on lysine, arginine, proline or threonine, or by secondary reaction of cysteine, histidine or lysine residues with carbonyl compounds can result in the formation of protein derivatives possessing highly reactive carbonyl groups such as aldehydes and ketones (Berlett et al., 1997). Oxidation of some critical methionine residues causes a complete inhibition of actin polymerization and destabilization of the structure of actin filaments (Dalle-Donne et al., 2002).

Oxidative modification of proteins by ROS is implicated in the etiology and/or progression of a broad variety of age-related degenerative pathologies, atherosclerosis, muscular dystrophy, rheumatoid arthritis (Chevion et al., 2000; Moreira et al., 2010; Stadtman &

Role of the Neutrophil NADPH Oxidase and

**2.2.1 Chronic inflammatory diseases**

S100A8/A9 in the Pathophysiology of Chronic Inflammation 311

production is a prominent feature in various cardiovascular-related diseases such as hypertension, atherosclerosis, and ischemic heart diseases. Moreover, it was suggested that ROS play a role in a variety of age-related diseases such as some neurological diseases like Alzheimer's disease, type 2 diabetes or even reproductive disorders (for reviews, see Kukreja & Hess, 1992; Reddy et al., 2009; Valko et al., 2007). Among this pleiad of diseases, ROS derived from the neutrophil NADPH oxidase complex (NOX2) have notably been implicated in the pathophysiology of rheumatoid arthritis, atherosclerosis, chronic

Rheumatoid arthritis is the most common type of rheumatological disorder with a prevalence of 0.4% to 0.7% worldwide (Alamanos et al., 2006; Englund et al., 2010). Rheumatoid arthritis is a chronic, destructive, autoimmune joint disease resulting in enormous pathologic sequelae including pain, stiffness, deformity, swelling, as well as systemic effects associated with inflammation limiting activities of daily living. Rheumatoid arthritis principally affects peripheral synovial joints but additionally extra-articular complications, including atherosclerotic vascular disease and premature mortality, can be associated to the disease (Carroll et al., 2006). Indeed, cardiovascular complications are the leading cause of death (42%) among patients with rheumatoid arthritis (Callahan et al., 1995). The pathogenesis of rheumatoid arthritis is a complex process with several distinguishing features involving macrophage-like synoviocytes and fibroblast-like

The presence of abundant numbers of neutrophils in the synovial fluid of patients with rheumatoid arthritis participate to joint damage via the release of potent effectors of cartilage destruction such as proteases and NOX2-mediated ROS production. The pathogenesis of rheumatoid arthritis is predominantly linked to the formation of ROS to the site of inflammation and tissue lesions caused by elevated ROS concentration participate to the perpetuation of inflammation. These oxidative derivatives may depolymerize hyaluronic acid and inactivate endogenous inhibitors of proteases (Chatham et al., 1993; Edwards & Hallett, 1997; Larbre et al., 1994; Robinson et al., 1992). Rheumatoid arthritis fluids can also contain large quantities of immune complexes and their deposition has been considered to be a major determinant of neutrophil-mediated destructive joint process which is characteristic of rheumatoid arthritis. Indeed, neutrophils primed and isolated from the synovial fluid of patients with rheumatoid arthritis can secrete substantial

Neutrophils have also been found at sites of atherosclerotic plaque rupture where they appear to be functionally active to generate ROS. Infiltrated neutrophils into atherosclerotic plaque underline the fact that these pro-inflammatory cells contribute to plaque

The inflammatory processes in the lung are characterized by an influx of neutrophils into the airways. Increased levels of NOX2-derived ROS produced by inflammatory cells of the airways in the lung have been recognized to contribute importantly in the pathogenesis of asthma and chronic obstructive pulmonary disease (Rahman, 2002). The enhanced expression of surface adhesion molecule Mac-1, which favours the recruitment of neutrophils to the inflammatory site, is coupled with an increase of NOX2 activity. It augments the potential of ROS to induce lung injury, contributing to the development of chronic obstructive pulmonary disease (Noguera et al., 2001). Oxidative stress can reduce

obstructive inflammatory disease and inflammation-associated cancer.

synoviocytes proliferation, pannus formation, cartilage and bone erosion.

quantities of ROS in response to immune complexes (Ottonello et al., 2002).

vulnerability and erosion (Hosokawa et al., 2011).

Berlett, 1997). Various human diseases have been associated with carbonylated proteins (Table 1): acute respiratory distress syndrome, Alzheimer's disease, rheumatoid arthritis, chronic lung disease, and diabetes (for review, see Dalle-Donne et al., 2003).

#### **2.1.3 Nucleic acid oxidation**

Permanent genetic material damage due to oxidative stress can represent the first step to mutagenesis, carcinogenesis, and ageing. ROS are known to react with all components of the DNA molecule and impair DNA repair mechanisms. DNA modification can affect both the purine and pyrimidine bases and also the deoxyribose backbone (Aruoma et al., 1989). DNA modification has been reported to accumulate over the life span of the cell participating to ageing (von Zglinicki et al., 1995). Telomere DNA is deficient in the repair of single-strand breaks produced by hydroxyl radicals affecting telomere length and hence active cell division (Oikawa et al., 2001; Petersen et al., 1998; Saretzki et al., 1999). The most extensively studied DNA lesion is the formation of 8-oxo-7-hydrodeoxyguanosine (8-OH-guanine) generated by the reaction of hydroxyl radicals with guanine repeats. 8-OH-guanine is particularly mutagenic and cytotoxic. It is elevated in leukocytes and sera of patients with rheumatoid arthritis (Table 1) and probably participates in joint inflammation by activating immune cells, which in turn produce excessive pro-inflammatory cytokines (Hajizadeh et al., 2003).

#### **2.1.4 Carbohydrate oxidation**

Glycosaminoglycans (GAGs) are major components of extracellular matrix and their increased synthesis and degradation are hallmarks of chronic inflammation and tissue fibrosis. GAGs determine the structure, viscosity and permeability of the ground substance in connective tissue and play a critical role in ion transport, nutrient diffusion, water homeostasis, intercellular signalling and collagen synthesis. GAG oxidation is known to have important biological consequences in particular on endothelial cell adhesion and proliferation (Underwood et al., 1998; Vissers et al., 1991) that are involved in atherogenesis. Chondroitin sulphates and hyaluronic acid, which contain glucosamine and glucuronic acid, are critical GAGs present in synovial joints that have been shown to be fragmented by ROS (Rees et al., 2004). The hypochlorite-mediated depolymerization of GAGs has been shown to produce polymer-derived chloramide, the major species formed due to the reaction of hypochlorite with glucosamine or galactosamine residues (Hawkins & Davis, 1998; Rees et al., 2003). Lower molecular weight hyaluronic acid fragments accumulate during inflammation and may have a role in the activation of phagocytes through the stimulation of interleukin-1 and TNF-α expression (Noble et al., 1993) resulting in an exacerbation of the inflammatory process and tissue damage.

#### **2.2 NOX2-derived ROS and inflammatory diseases**

Chronic inflammation was hypothesized as the loss of balance between apoptosis and wound healing leading to disruption of protective mechanisms of immune system (Khatami, 2005, 2008, 2009). Unresolved inflammation-induced excessive expression of proand anti-inflammatory mediators causes erosion of tissue integrity initiating the development of chronic inflammatory diseases or cancer (Khatami, 2011).

Molecular alterations, induced by exaggerated ROS generation during oxidative stress, represent an important cause of injury in many inflammatory diseases (Table 1). ROS production is a prominent feature in various cardiovascular-related diseases such as hypertension, atherosclerosis, and ischemic heart diseases. Moreover, it was suggested that ROS play a role in a variety of age-related diseases such as some neurological diseases like Alzheimer's disease, type 2 diabetes or even reproductive disorders (for reviews, see Kukreja & Hess, 1992; Reddy et al., 2009; Valko et al., 2007). Among this pleiad of diseases, ROS derived from the neutrophil NADPH oxidase complex (NOX2) have notably been implicated in the pathophysiology of rheumatoid arthritis, atherosclerosis, chronic obstructive inflammatory disease and inflammation-associated cancer.

#### **2.2.1 Chronic inflammatory diseases**

Inflammation, Chronic Diseases and Cancer –

310 Cell and Molecular Biology, Immunology and Clinical Bases

Berlett, 1997). Various human diseases have been associated with carbonylated proteins (Table 1): acute respiratory distress syndrome, Alzheimer's disease, rheumatoid arthritis,

Permanent genetic material damage due to oxidative stress can represent the first step to mutagenesis, carcinogenesis, and ageing. ROS are known to react with all components of the DNA molecule and impair DNA repair mechanisms. DNA modification can affect both the purine and pyrimidine bases and also the deoxyribose backbone (Aruoma et al., 1989). DNA modification has been reported to accumulate over the life span of the cell participating to ageing (von Zglinicki et al., 1995). Telomere DNA is deficient in the repair of single-strand breaks produced by hydroxyl radicals affecting telomere length and hence active cell division (Oikawa et al., 2001; Petersen et al., 1998; Saretzki et al., 1999). The most extensively studied DNA lesion is the formation of 8-oxo-7-hydrodeoxyguanosine (8-OH-guanine) generated by the reaction of hydroxyl radicals with guanine repeats. 8-OH-guanine is particularly mutagenic and cytotoxic. It is elevated in leukocytes and sera of patients with rheumatoid arthritis (Table 1) and probably participates in joint inflammation by activating immune cells, which in turn produce

Glycosaminoglycans (GAGs) are major components of extracellular matrix and their increased synthesis and degradation are hallmarks of chronic inflammation and tissue fibrosis. GAGs determine the structure, viscosity and permeability of the ground substance in connective tissue and play a critical role in ion transport, nutrient diffusion, water homeostasis, intercellular signalling and collagen synthesis. GAG oxidation is known to have important biological consequences in particular on endothelial cell adhesion and proliferation (Underwood et al., 1998; Vissers et al., 1991) that are involved in atherogenesis. Chondroitin sulphates and hyaluronic acid, which contain glucosamine and glucuronic acid, are critical GAGs present in synovial joints that have been shown to be fragmented by ROS (Rees et al., 2004). The hypochlorite-mediated depolymerization of GAGs has been shown to produce polymer-derived chloramide, the major species formed due to the reaction of hypochlorite with glucosamine or galactosamine residues (Hawkins & Davis, 1998; Rees et al., 2003). Lower molecular weight hyaluronic acid fragments accumulate during inflammation and may have a role in the activation of phagocytes through the stimulation of interleukin-1 and TNF-α expression (Noble et al., 1993) resulting in an exacerbation of the

Chronic inflammation was hypothesized as the loss of balance between apoptosis and wound healing leading to disruption of protective mechanisms of immune system (Khatami, 2005, 2008, 2009). Unresolved inflammation-induced excessive expression of proand anti-inflammatory mediators causes erosion of tissue integrity initiating the

Molecular alterations, induced by exaggerated ROS generation during oxidative stress, represent an important cause of injury in many inflammatory diseases (Table 1). ROS

development of chronic inflammatory diseases or cancer (Khatami, 2011).

chronic lung disease, and diabetes (for review, see Dalle-Donne et al., 2003).

excessive pro-inflammatory cytokines (Hajizadeh et al., 2003).

**2.1.3 Nucleic acid oxidation**

**2.1.4 Carbohydrate oxidation**

inflammatory process and tissue damage.

**2.2 NOX2-derived ROS and inflammatory diseases**

Rheumatoid arthritis is the most common type of rheumatological disorder with a prevalence of 0.4% to 0.7% worldwide (Alamanos et al., 2006; Englund et al., 2010). Rheumatoid arthritis is a chronic, destructive, autoimmune joint disease resulting in enormous pathologic sequelae including pain, stiffness, deformity, swelling, as well as systemic effects associated with inflammation limiting activities of daily living. Rheumatoid arthritis principally affects peripheral synovial joints but additionally extra-articular complications, including atherosclerotic vascular disease and premature mortality, can be associated to the disease (Carroll et al., 2006). Indeed, cardiovascular complications are the leading cause of death (42%) among patients with rheumatoid arthritis (Callahan et al., 1995). The pathogenesis of rheumatoid arthritis is a complex process with several distinguishing features involving macrophage-like synoviocytes and fibroblast-like synoviocytes proliferation, pannus formation, cartilage and bone erosion.

The presence of abundant numbers of neutrophils in the synovial fluid of patients with rheumatoid arthritis participate to joint damage via the release of potent effectors of cartilage destruction such as proteases and NOX2-mediated ROS production. The pathogenesis of rheumatoid arthritis is predominantly linked to the formation of ROS to the site of inflammation and tissue lesions caused by elevated ROS concentration participate to the perpetuation of inflammation. These oxidative derivatives may depolymerize hyaluronic acid and inactivate endogenous inhibitors of proteases (Chatham et al., 1993; Edwards & Hallett, 1997; Larbre et al., 1994; Robinson et al., 1992). Rheumatoid arthritis fluids can also contain large quantities of immune complexes and their deposition has been considered to be a major determinant of neutrophil-mediated destructive joint process which is characteristic of rheumatoid arthritis. Indeed, neutrophils primed and isolated from the synovial fluid of patients with rheumatoid arthritis can secrete substantial quantities of ROS in response to immune complexes (Ottonello et al., 2002).

Neutrophils have also been found at sites of atherosclerotic plaque rupture where they appear to be functionally active to generate ROS. Infiltrated neutrophils into atherosclerotic plaque underline the fact that these pro-inflammatory cells contribute to plaque vulnerability and erosion (Hosokawa et al., 2011).

The inflammatory processes in the lung are characterized by an influx of neutrophils into the airways. Increased levels of NOX2-derived ROS produced by inflammatory cells of the airways in the lung have been recognized to contribute importantly in the pathogenesis of asthma and chronic obstructive pulmonary disease (Rahman, 2002). The enhanced expression of surface adhesion molecule Mac-1, which favours the recruitment of neutrophils to the inflammatory site, is coupled with an increase of NOX2 activity. It augments the potential of ROS to induce lung injury, contributing to the development of chronic obstructive pulmonary disease (Noguera et al., 2001). Oxidative stress can reduce

Role of the Neutrophil NADPH Oxidase and

Source Targets/alterations

ROS Lipid peroxidation

ROS Carbonylated proteins

NF-κB, p38 MAPK activation

p38 MAPK, p44/42 kinase activation

Transcription factor activation

Hyaluronic acid depolymerization Protease inhibitor inactivation

Atherosclerotic plaque erosion

p38 MAPK, EGFR, SHP2 modulation

ROS DNA lesions Lung cancer

S100A8/A9 Rheumatoid arthritis

Table 1. Some human inflammatory diseases associated with ROS and S100A8/A9

**3. Description of the signal transduction pathways regulating NOX2 with**

Ca2+ is a ubiquitous second messenger that functions over a wide and defined spatiotemporal range. Normal cytosolic free Ca2+ concentration ([Ca2+]c) in resting mammalian cells is maintained to low levels, in a range of 50-150 nM, compared to an endoplasmic reticulum and extracellular Ca2+ concentration of 500 M and 2000 M, respectively. When Ca2+ channels are opened in the plasma membrane, Ca2+ can rapidly flows into the cytosol and reach a concentration of 1 M in a few seconds. Alternatively, Ca2+ influx can be delivered to low sustained Ca2+ levels to control appropriate cellular activities. A broad class of Ca2+ channels, differing for regulatory mechanisms and intracellular distribution, can be activated allowing the control by adapted Ca2+-signalling systems of many divergent cellular processes (exocytosis, muscle contraction, gene transcription, fertilization, meiosis, immune response, muscle contraction) with the right frequency and intensity. Fluctuations in [Ca2+]c are initiated at a localized site and diffuse inside the cell in the form of intracellular Ca2+ waves. In this view, neutrophils show irregular [Ca2+]c spikes during phagocytosis and after stimulation by the bacterial chemopeptide fMLF (Dewitt et al., 2006; Jaconi et al., 1988).

The receptor-mediated Ca2+ influx required for NOX2 activation in neutrophils is predominantly the result of the activation of the so-called store-operated Ca2+ entry (SOCE), which is initiated by a fall in intracellular Ca2+ store content. The store depletion is coupled to the opening of store-operated Ca2+ channels (SOCs) localized in the plasma membrane,

ROS Histone deacetylase activity reduction

S100A8/A9 Myeloid-derived suppressor cell accumulation

**emphasis on the role of Ca2+ mobilization**

**3.1 Store-operated Ca2+ entry mechanism**

ROS

S100A8/A9 in the Pathophysiology of Chronic Inflammation 313

Nucleic acid oxidation Rheumatoid arthritis

Diabetes, Alzheimer's disease

Atherosclerosis

Cancer

Chronic obstructive pulmonary disease

the histone deacetylase activity allowing the access of transcription factors as NF-κB, AP-1 and Nrf2 to the DNA and leading to enhanced pro-inflammatory or reduced antioxidant gene expression in various lung cells (Ito et al., 2001; Kersul et al., 2001; Mercado et al., 2011). Moreover, ROS may play a role in enhancing the mucus secretion through the modulation of enzymatic activities such as src homology 2-containing protein tyrosine phosphatase, p38 MAPK or receptor tyrosine kinases (epidermal growth factor receptor) (Jang et al., 2010; Kohri et al., 2002; Meng et al., 2002).

#### **2.2.2 Cancer-associated inflammation**

Persistent chronic inflammation contributes to increase the risk of cancer and promote cancer development and progression. The inflammatory response triggered by infection is involved in the pathogenesis of approximately 20% of human tumors (e.g., Helicobacter pylori infection is associated with gastric cancer). Cancer-related inflammation is characterized by the presence of infiltrating immune and inflammatory cells (notably Tlymphocytes, tumor-associated macrophages and neutrophils) in the microenvironment of the tumor increasing generation of ROS and angiogenic factors, matrix-degrading enzyme and growth factor activities, and altering cytokine and chemokine expression. Accumulating evidence shows that chronic inflammation can promote an environment that is favourable to all the stages of human tumors. Six hallmarks have been proposed by Hanahan & Weinberg (Hanahan & Weinberg, 2011) to characterize the multistep of the carcinogenesis including sustaining proliferative signalling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis. It has been proposed that the genome instability leading to cancer-related inflammation represents the seventh hallmark of tumorigenesis (Allavena et al., 2008; Mantovani, 2010).

The link between chronic inflammation and cancer has been suggested by the enhanced colorectal cancer susceptibility of persons with inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease). Repeated injury and repair triggered by chronic inflammation may increase cell turnover and permanent changes in the genome leading ultimately to tumorigenesis. It is generally accepted that cancer initiation may be a pathological consequence of ROS-induced DNA lesions that in turn result in mutations activating oncogenes or inactivating tumor-suppressor genes (Klaunig & Kamendulis, 2004). Chronic exposure to ROS may also alter normal cellular and molecular signalling pathways. ROS have been shown to activate transcription factors such NF-κB, AP-1 and STATs. NF-κB is known to be constitutively activated in a variety of malignancies (Brar et al., 2003) contributing to abnormal malignant cell division through enhanced expression of anti-apoptotic factors (Bcl-2), and proliferative factors, such as cyclin D1 (Brar et al., 2003). The interest in the role of neutrophils in the inflammatory origin of cancer (Table 1) is recent and has considerably increased over the last years. In addition of its function in host defence, a critical role for NOX2-mediated ROS production has been pointed in the induction and development of malignant cells. Human neutrophils can directly damage DNA and can induce malignant transformation, which suggest that phagocytic cells are carcinogenic (Brar et al., 2003; Knaapen et al., 1999). The leukotriene LTB-4 and cytokine IL-8 are recognized to play a crucial role in neutrophil recruitment into airways during lung cancer in particular when progressing to the next stage of cancer (Carpagnano et al., 2011).

312 Cell and Molecular Biology, Immunology and Clinical Bases

the histone deacetylase activity allowing the access of transcription factors as NF-κB, AP-1 and Nrf2 to the DNA and leading to enhanced pro-inflammatory or reduced antioxidant gene expression in various lung cells (Ito et al., 2001; Kersul et al., 2001; Mercado et al., 2011). Moreover, ROS may play a role in enhancing the mucus secretion through the modulation of enzymatic activities such as src homology 2-containing protein tyrosine phosphatase, p38 MAPK or receptor tyrosine kinases (epidermal growth factor receptor)

Persistent chronic inflammation contributes to increase the risk of cancer and promote cancer development and progression. The inflammatory response triggered by infection is involved in the pathogenesis of approximately 20% of human tumors (e.g., Helicobacter pylori infection is associated with gastric cancer). Cancer-related inflammation is characterized by the presence of infiltrating immune and inflammatory cells (notably Tlymphocytes, tumor-associated macrophages and neutrophils) in the microenvironment of the tumor increasing generation of ROS and angiogenic factors, matrix-degrading enzyme and growth factor activities, and altering cytokine and chemokine expression. Accumulating evidence shows that chronic inflammation can promote an environment that is favourable to all the stages of human tumors. Six hallmarks have been proposed by Hanahan & Weinberg (Hanahan & Weinberg, 2011) to characterize the multistep of the carcinogenesis including sustaining proliferative signalling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis. It has been proposed that the genome instability leading to cancer-related inflammation represents the seventh hallmark of tumorigenesis (Allavena et al., 2008;

The link between chronic inflammation and cancer has been suggested by the enhanced colorectal cancer susceptibility of persons with inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease). Repeated injury and repair triggered by chronic inflammation may increase cell turnover and permanent changes in the genome leading ultimately to tumorigenesis. It is generally accepted that cancer initiation may be a pathological consequence of ROS-induced DNA lesions that in turn result in mutations activating oncogenes or inactivating tumor-suppressor genes (Klaunig & Kamendulis, 2004). Chronic exposure to ROS may also alter normal cellular and molecular signalling pathways. ROS have been shown to activate transcription factors such NF-κB, AP-1 and STATs. NF-κB is known to be constitutively activated in a variety of malignancies (Brar et al., 2003) contributing to abnormal malignant cell division through enhanced expression of anti-apoptotic factors (Bcl-2), and proliferative factors, such as cyclin D1 (Brar et al., 2003). The interest in the role of neutrophils in the inflammatory origin of cancer (Table 1) is recent and has considerably increased over the last years. In addition of its function in host defence, a critical role for NOX2-mediated ROS production has been pointed in the induction and development of malignant cells. Human neutrophils can directly damage DNA and can induce malignant transformation, which suggest that phagocytic cells are carcinogenic (Brar et al., 2003; Knaapen et al., 1999). The leukotriene LTB-4 and cytokine IL-8 are recognized to play a crucial role in neutrophil recruitment into airways during lung cancer in particular when progressing to the next stage of

(Jang et al., 2010; Kohri et al., 2002; Meng et al., 2002).

**2.2.2 Cancer-associated inflammation**

Mantovani, 2010).

cancer (Carpagnano et al., 2011).

Table 1. Some human inflammatory diseases associated with ROS and S100A8/A9

#### **3. Description of the signal transduction pathways regulating NOX2 with emphasis on the role of Ca2+ mobilization**

Ca2+ is a ubiquitous second messenger that functions over a wide and defined spatiotemporal range. Normal cytosolic free Ca2+ concentration ([Ca2+]c) in resting mammalian cells is maintained to low levels, in a range of 50-150 nM, compared to an endoplasmic reticulum and extracellular Ca2+ concentration of 500 M and 2000 M, respectively. When Ca2+ channels are opened in the plasma membrane, Ca2+ can rapidly flows into the cytosol and reach a concentration of 1 M in a few seconds. Alternatively, Ca2+ influx can be delivered to low sustained Ca2+ levels to control appropriate cellular activities. A broad class of Ca2+ channels, differing for regulatory mechanisms and intracellular distribution, can be activated allowing the control by adapted Ca2+-signalling systems of many divergent cellular processes (exocytosis, muscle contraction, gene transcription, fertilization, meiosis, immune response, muscle contraction) with the right frequency and intensity. Fluctuations in [Ca2+]c are initiated at a localized site and diffuse inside the cell in the form of intracellular Ca2+ waves. In this view, neutrophils show irregular [Ca2+]c spikes during phagocytosis and after stimulation by the bacterial chemopeptide fMLF (Dewitt et al., 2006; Jaconi et al., 1988).

#### **3.1 Store-operated Ca2+ entry mechanism**

The receptor-mediated Ca2+ influx required for NOX2 activation in neutrophils is predominantly the result of the activation of the so-called store-operated Ca2+ entry (SOCE), which is initiated by a fall in intracellular Ca2+ store content. The store depletion is coupled to the opening of store-operated Ca2+ channels (SOCs) localized in the plasma membrane,

Role of the Neutrophil NADPH Oxidase and

cytosolic Ca2+ (Brasen et al., 2011).

(Braun et al., 2009; Steinckwich et al., 2011).

regulation of NOX2 (Bréchard et al., 2008).

**3.4.1 Protein kinase C (PKC)**

or SOCE and NOX2 has been yet formally established.

**3.4 Ca2+ targets for the regulation of NOX2 activity**

bind to either diacylglycerol or phorbol esters.

enzymes for a full activation of NOX2 (Korchak et al., 2007).

S100A8/A9 in the Pathophysiology of Chronic Inflammation 315

2008; Foyouzi-Youssefi et al., 1997). Knock-down of putative constituents of SOCE mechanism (Orai1, STIM1) by specific siRNA or decrease of SOC activity by pharmacological inhibitors (MRS1845, SK&F96365 or 2-APB) led to a diminished NOX2 activity in the plasma membrane (Bréchard et al., 2008, 2009; Lee et al., 2005) underlining the preponderant function of SOCE in the regulation of NOX2. Activation of Ca2+ signal is reported as an on-switch for NOX2 activity not directly correlated with spiking activity in

Extracellular Ca2+ entry is also required for intraphagosomal oxidative activation and has been temporally correlated with NOX2 activity (Dewitt et al., 2002). Ca2+ influx appears to be necessary for an optimal intraphagosomal oxidative activity but not sufficient to initiate oxidative activation (Dewitt et al., 2002). Orai1 and STIM1 contribute to the regulation of phagosomal NOX2 activity through the activation of store depletion-regulated Ca2+ influx

On the other hand, Itagaki et al. (Itagaki et al., 2005) suggest that Ca2+ influx occurring through a mechanism other than SOCE could be a relevant event to activate NOX2. In addition, the TRP channel, TRPC3, appears to be involved in non-SOCE-dependent