quirements of Treg activation and inactivation are still to be determined.

**3. Impact of CD86/CD80 molecules in autoimmune disease**

ment and maintenance of Treg cells [58].

104 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

Systemic lupus erythematosus (SLE) is a chronic multi-organ autoimmune disease distin‐ guish by imbalanced T-cell homeostasis towards activated Teff cell subsets Th1 and Th2 and can affect multiple organs and systems of the body. It is well characterized by the number and variety of autoantibodies produced and is well marked by anti-nuclear anti‐ body production. Many of the symptoms are caused by impaired antigen-antibody com‐ plex clearance and thus triggering inflammatory responses in multiple sites. Because of the difficulty in evaluating patient disease progression several mouse models have been used to evaluate how abnormal costimulation and Treg cells contribute to disease. The autoimmune BXD2 mice indicates that type I IFN can promote autoimmune responses through the upregulation of CD86high expression on marginal zone precursor B-cells, which were shown to be located at the T-B-cell border of the spleen germinal centers [60]. In regards to Treg maintenance, the (NZB x NZW)F1 and (SWR/NZB)F1 lupusprone mice had reduced numbers of CD4+CD25+ cells [61]. There was no intrinsic defect in the suppressive function of the (NZB x NZW) F1 mice [62]. Additionally, only margin‐ al defects in Treg suppression were observed in MRL/Mp mice [63]. MRL/lpr mice with strong lupus disease were found to have normal percentages of peripheral CD4+CD25high T-cells and that Foxp3 expression was unaltered. However, they do dis‐ play a reduced capacity to suppress and effector CD4+CD25- T-cells were significantly less susceptible to suppression. Importantly they also found that CD80/CD86 were under‐ expressed on Teff, APCs, and on Treg cells, suggesting that the reduction in these mole‐ cules could be contributing to the reduced abilities of Treg cells to suppress [64].

#### **3.2. Rheumatoid arthritis**

Rheumatoid Arthritis (RA) is a chronic inflammatory disease characterized by inflammation of the joints and surrounding tissue including mononuclear cell infiltration of synovial tis‐ sue. Predominant lymphocytes present in rheumatoid synovium focal infiltrates are lym‐ phocytes (predominantly CD4+ T-cells with relatively few positive for CD25), macrophages, and plasma B cells. The expression of CD86 was readily detectable in the synovium com‐ pared to osteoarthritis synovial [65]. In contrast, CD80 expression was not significantly ex‐ pressed in the synovium [65]. Overall, the expression of CD80 on APCs in the synovium is generally low, while expression of CD86 is relatively high and is expressed on several APCs including DCs, B-cells, and macrophages [66-70]. Blocking of CD28 signaling pathway has also been shown to prevent or even treat autoimmune disease [71], indicating that CD28 is probably a regulator in the induction of autoimmune diseases. For instance, CD28 deficient mice are resistant to collagen induced arthritis [72] and inhibition of both CD80 and CD86 during the induction phase of collagen induced arthritis prevents the development of dis‐ ease [71, 73, 74]. Arthritis is also abolished and autoantibody production is suppressed in MLR/lpr mice lacking CD28, however, the accumulation of abnormal T cells is almost un‐ changed [75]. Indicating CD28 may have a better role in aiding B cell responses in this mod‐ el. Most importantly, treatment with blocking CTLA-4-Ig also significantly improved signs and symptoms and Treg function of RA patients in clinical trials [76]. In general, Treg cells with diminished suppressive capacity were obtained from synovial fluid of patients with RA [77, 78]. Functional studies of Treg cell defects in mice related to RA indicate that defects in CD4+Foxp3+ Treg cells in K/BxNsf mice exhibited earlier onset and more aggressive pro‐ gression of arthritis than K/BxN littermates [79]. This was accompanied by plasmacytoid dendritic cells expressing high levels of CD86 and CD40, but not CD80, in synovia and in‐ crease memory CD4+ T-cells in the spleen and draining lymph nodes [79]. These mice also exhibit an abnormal accumulation of mature plasma cells in spleen and associated loss of bone marrow plasma cells. These plasma cells were also less susceptible to cell death [80]. Overall, it appears the function of CD28 to the pathogenesis of RA, potentially mediated through over expression of CD86 appears to have a major function in disease pathogenesis.

[53, 54]. The importance of Treg number to quality of suppression was shown with polyclo‐ nal Treg adoptive transfer to NOD.scid at a 2:1 ratio (Treg:Th1) was unable to suppress, while 5:1 ratio was able to provide protection in approximately half of the recipients. There‐ fore, with sufficiently large populations of Tregs, there may be adequate numbers of anti‐ gen-specific Tregs capable of suppressing diabetes [85]. It is interesting to note that Treg cells developed in vitro from induced GAD-IgG transduced splenocytes were capable of suppressing diabetes in NOD mice. This was accompanied by a higher ratio of CD80 com‐ pared to CD86 expression in splenocytes, which was sufficient to allow development of functional Treg cells (Foxp3+) in this model [86-88]. Depletion of CD4+CD25+ cells from transduced splenocytes transferred into NOD mice showed increased ratio of CD86 to CD80 in splenocytes. Blocking of CD80, but not CD86, reduced the relative quantity of Foxp3 [87]. Along these lines, a progressive decrease was observed in the Treg cell:Teff cell ratio in in‐ flamed islets but not in pancreatic lymph nodes in NOD mice. Intra-islet Treg cells ex‐ pressed reduced amounts of CD25 and Bcl-2, where the administration of low-dose interleukin-2 (IL-2) promoted Treg cell survival and protected mice from developing diabe‐ tes. Together, these results suggest intra-islet Treg cell dysfunction is a root cause of the pro‐ gressive breakdown of self-tolerance and the development of diabetes in nonobese diabetic mice [89]. However, there is still some debate on whether NOD mouse Treg cells are suffi‐ cient in regulating Teff, since Tregs from NOD and B6g7 mice were equally effective but

Costimulatory Molecules in Rheumatic Diseases Revisited with an Emphasis on Their Roles in…

http://dx.doi.org/10.5772/54480

107

Recently in SjS, as with some of the other autoimmune diseases previously mentioned, the emphasis of pathogenicity has been placed on CD86 expression in the salivary gland envi‐ ronment. Expression of CD86 and CD80 in SjS patient salivary gland tissue compared to pa‐ tients with nonspecific sialadenitis was first described in 1999, where expression of both ligands were found in ductal and acinar regions of immunohistochemically stained tissue sections [91]. It was recently shown that the functional expression of CD86 on salivary gland epithelial cells derived from SjS patients are capable of interacting with CD28, but its bind‐ ing to CTLA-4 was reduced [92]. Therefore, salivary epithelial cells are possibly functioning to promote production of IL-2 and T-cell proliferation. This paper also indicates that expres‐ sion of CD80, contrary to other immunohistochemistry results suggesting that CD80 is ex‐ pressed [93],[94], could not be established in their salivary gland epithelial cells. It is difficult to establish the relative expression of these molecules in vivo, since several other studies were conducted on cell lines derived from patient biopsies under cytokine stimulating con‐ ditions or from immunohistochemistry of tissue sections using BB1 antibody, which poten‐ tially shares reactivity with non-B7 proteins such as MHC class II invariant chain [92, 95]. Further studies may be required to verify the production of CD80 as well as the relative ex‐ pression of each of these ligands in the salivary glands of patients and healthy controls.

However, in C57BL/6 (B6) mice the overall expression of B7 is relatively low (CD86 greater than CD80) in normal mouse submandibular salivary glands compared to lymphoid tissues and is located predominantly along the ducts and among acini as previously indicated in

NOD T conventional cells were hyper-responsive to stimulation [90].

**3.4. Sjögren's syndrome**

#### **3.3. Autoimmune diabetes**

B7 molecules have potential influence on autoreactive T cells in animals genetically pre‐ disposed to autoimmune disease. Adoptive transfer of NOD B-cells previously blocked with B7 MAbs along with diabetogenic T-cells into NOD.scid mice protected associated type 1 diabetes [81].Again, as mentioned previously NOD mice treated with blocking CD86 MAbs prevents the spontaneous development of diabetes, whereas blocking CD80 accelerates and worsened disease [48]. Treatment of NOD mice older than 10 weeks of age with blocking CD86 MAbs did not alter the course of disease [48]. These results indi‐ cate that in the NOD background, the influence of CD86 to promotion and CD80 to sup‐ pression of disease occurs early on in disease pathogenesis. Breeding of CD86 deficiency onto NOD background prevented the development of diabetes, as expected from previ‐ ous studies. However, aging mice around age 20 weeks would develop a peripheral neu‐ ropathy characterized by demyelization and defective nerve function due to mononuclear cell infiltration of peripheral nerves [82]. This was accompanied by a high level of CD80 expression on the APCs in the spleen as well as on the nerves of affected animals [82]. This is similar to reports seen in Experimental Autoimmune Epithelitis (EAE) and Multi‐ ple Sclerosis (MS) patients [83, 84], where downregulation of CD86 and upregulation of CD80 is observed on CNS-infiltrating cells and splenocytes. These somewhat conflicting findings for autoimmune diseases can potentially be explained by differences in interplay of local cellular interactions, cytokines and chemokines that may alter B7 expression. In this case, high expression of CD80 could potentially allow activation of CD28 signaling and autoimmune activation rather than suppression.

There is still some debate on whether Treg cells contribute to autoimmunity with regards to their deficits and functionality in autoimmune diabetes. Treg depletion or B7-deficiency (sig‐ nificant loss in CD4+CD25+ cell population) in NOD mice leads to accelerated disease onset [53, 54]. The importance of Treg number to quality of suppression was shown with polyclo‐ nal Treg adoptive transfer to NOD.scid at a 2:1 ratio (Treg:Th1) was unable to suppress, while 5:1 ratio was able to provide protection in approximately half of the recipients. There‐ fore, with sufficiently large populations of Tregs, there may be adequate numbers of anti‐ gen-specific Tregs capable of suppressing diabetes [85]. It is interesting to note that Treg cells developed in vitro from induced GAD-IgG transduced splenocytes were capable of suppressing diabetes in NOD mice. This was accompanied by a higher ratio of CD80 com‐ pared to CD86 expression in splenocytes, which was sufficient to allow development of functional Treg cells (Foxp3+) in this model [86-88]. Depletion of CD4+CD25+ cells from transduced splenocytes transferred into NOD mice showed increased ratio of CD86 to CD80 in splenocytes. Blocking of CD80, but not CD86, reduced the relative quantity of Foxp3 [87]. Along these lines, a progressive decrease was observed in the Treg cell:Teff cell ratio in in‐ flamed islets but not in pancreatic lymph nodes in NOD mice. Intra-islet Treg cells ex‐ pressed reduced amounts of CD25 and Bcl-2, where the administration of low-dose interleukin-2 (IL-2) promoted Treg cell survival and protected mice from developing diabe‐ tes. Together, these results suggest intra-islet Treg cell dysfunction is a root cause of the pro‐ gressive breakdown of self-tolerance and the development of diabetes in nonobese diabetic mice [89]. However, there is still some debate on whether NOD mouse Treg cells are suffi‐ cient in regulating Teff, since Tregs from NOD and B6g7 mice were equally effective but NOD T conventional cells were hyper-responsive to stimulation [90].

#### **3.4. Sjögren's syndrome**

during the induction phase of collagen induced arthritis prevents the development of dis‐ ease [71, 73, 74]. Arthritis is also abolished and autoantibody production is suppressed in MLR/lpr mice lacking CD28, however, the accumulation of abnormal T cells is almost un‐ changed [75]. Indicating CD28 may have a better role in aiding B cell responses in this mod‐ el. Most importantly, treatment with blocking CTLA-4-Ig also significantly improved signs and symptoms and Treg function of RA patients in clinical trials [76]. In general, Treg cells with diminished suppressive capacity were obtained from synovial fluid of patients with RA [77, 78]. Functional studies of Treg cell defects in mice related to RA indicate that defects in CD4+Foxp3+ Treg cells in K/BxNsf mice exhibited earlier onset and more aggressive pro‐ gression of arthritis than K/BxN littermates [79]. This was accompanied by plasmacytoid dendritic cells expressing high levels of CD86 and CD40, but not CD80, in synovia and in‐ crease memory CD4+ T-cells in the spleen and draining lymph nodes [79]. These mice also exhibit an abnormal accumulation of mature plasma cells in spleen and associated loss of bone marrow plasma cells. These plasma cells were also less susceptible to cell death [80]. Overall, it appears the function of CD28 to the pathogenesis of RA, potentially mediated through over expression of CD86 appears to have a major function in disease pathogenesis.

106 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

B7 molecules have potential influence on autoreactive T cells in animals genetically pre‐ disposed to autoimmune disease. Adoptive transfer of NOD B-cells previously blocked with B7 MAbs along with diabetogenic T-cells into NOD.scid mice protected associated type 1 diabetes [81].Again, as mentioned previously NOD mice treated with blocking CD86 MAbs prevents the spontaneous development of diabetes, whereas blocking CD80 accelerates and worsened disease [48]. Treatment of NOD mice older than 10 weeks of age with blocking CD86 MAbs did not alter the course of disease [48]. These results indi‐ cate that in the NOD background, the influence of CD86 to promotion and CD80 to sup‐ pression of disease occurs early on in disease pathogenesis. Breeding of CD86 deficiency onto NOD background prevented the development of diabetes, as expected from previ‐ ous studies. However, aging mice around age 20 weeks would develop a peripheral neu‐ ropathy characterized by demyelization and defective nerve function due to mononuclear cell infiltration of peripheral nerves [82]. This was accompanied by a high level of CD80 expression on the APCs in the spleen as well as on the nerves of affected animals [82]. This is similar to reports seen in Experimental Autoimmune Epithelitis (EAE) and Multi‐ ple Sclerosis (MS) patients [83, 84], where downregulation of CD86 and upregulation of CD80 is observed on CNS-infiltrating cells and splenocytes. These somewhat conflicting findings for autoimmune diseases can potentially be explained by differences in interplay of local cellular interactions, cytokines and chemokines that may alter B7 expression. In this case, high expression of CD80 could potentially allow activation of CD28 signaling

There is still some debate on whether Treg cells contribute to autoimmunity with regards to their deficits and functionality in autoimmune diabetes. Treg depletion or B7-deficiency (sig‐ nificant loss in CD4+CD25+ cell population) in NOD mice leads to accelerated disease onset

**3.3. Autoimmune diabetes**

and autoimmune activation rather than suppression.

Recently in SjS, as with some of the other autoimmune diseases previously mentioned, the emphasis of pathogenicity has been placed on CD86 expression in the salivary gland envi‐ ronment. Expression of CD86 and CD80 in SjS patient salivary gland tissue compared to pa‐ tients with nonspecific sialadenitis was first described in 1999, where expression of both ligands were found in ductal and acinar regions of immunohistochemically stained tissue sections [91]. It was recently shown that the functional expression of CD86 on salivary gland epithelial cells derived from SjS patients are capable of interacting with CD28, but its bind‐ ing to CTLA-4 was reduced [92]. Therefore, salivary epithelial cells are possibly functioning to promote production of IL-2 and T-cell proliferation. This paper also indicates that expres‐ sion of CD80, contrary to other immunohistochemistry results suggesting that CD80 is ex‐ pressed [93],[94], could not be established in their salivary gland epithelial cells. It is difficult to establish the relative expression of these molecules in vivo, since several other studies were conducted on cell lines derived from patient biopsies under cytokine stimulating con‐ ditions or from immunohistochemistry of tissue sections using BB1 antibody, which poten‐ tially shares reactivity with non-B7 proteins such as MHC class II invariant chain [92, 95]. Further studies may be required to verify the production of CD80 as well as the relative ex‐ pression of each of these ligands in the salivary glands of patients and healthy controls.

However, in C57BL/6 (B6) mice the overall expression of B7 is relatively low (CD86 greater than CD80) in normal mouse submandibular salivary glands compared to lymphoid tissues and is located predominantly along the ducts and among acini as previously indicated in human patients (data unpublished, [96]). Presumably, these cells are salivary gland epithe‐ lial cells, where the expression of low level costimulatory ligands is presumed normal and possibly protective. As for mouse models for SjS, retinoblastoma-associated protein 48 (RbAp48) transgenic mice, using a salivary gland specific promoter [97], resulted in the de‐ velopment of SjS-like symptoms. In salivary gland tissues of these mice the protein expres‐ sion of MHC class II, CD86, CD80, and ICAM-1 were all upregulated in affected mice salivary glands. Where the expression levels of CD86 is higher than CD80 in the SMX, indi‐ cating a potential pathogenic role of CD86 to disease initiation [96]. Another mouse model of SjS, the C57BL/6.NOD-*Aec1Aec2* is a double congenic mouse model of primary SjS syn‐ drome that contains two genetic regions (Idd3 and Idd5) derived from the NOD mouse model [98]. These genetic regions confer spontaneous development of SjS-like syndrome on the B6 background. A recent study using this model highlighted the importance of local B7 molecules and signaling through CD28 to disease progression [99]. In this report AAV trans‐ duced expression of the CTLA-4-Ig (blocks B7 molecules interactions) in the salivary glands of SjS-prone mouse model. Delivery of this gene construct via AAV prior to disease onset prevented glandular damage and lymphocytic cell infiltration commonly associated with disease, as well as, prevented loss of saliva secretion in the treated mice [99]. This was also accompanied by a significant increase in transforming growth factor beta-1 (TGF-B1) in the salivary glands and draining lymph nodes of these mice [99]. These results suggest a poten‐ tial regulatory role (either Treg or epithelial cells) involved with treatment of CTLA-4-Ig. Authors of this paper did not explore the mechanisms of action of this molecule. Along simi‐ lar lines, another study involving treatment of blocking CD86 MAbs to another mouse mod‐ el of primary SjS, the NFS/sld 3-day thymectomized mutant, was shown to prevent the autoimmune lesions and autoantibody production to a-fodrin [100]. Along with previous lines of research indicating a negative regulatory role of CD80, no significant effects were seen in mice treated with anti-CD80 MAbs [100]. These results together outline the impor‐ tance of costimulation to disease onset and severity and highlight the influence of CD86- CD28 as a potential mediator of disease initiation in salivary and lacrimal glands.

SjS patients have also been shown to have decreased number of Tregs (CCR7+Foxp3+) in salivary glands compared to controls [101]. In line with this direct tissue evidence, there are also several other reports of fewer salivary Tregs present in patients with SjS [103, 104] and reports of Tregs with decreased suppression capabilities [105]. Reports of increased number of Foxp3+ Treg cells in the salivary glands of patients with SjS [105, 106], was positively cor‐ related with the grade of infiltration as evaluated by Chisholm score [106]. The authors do not address potential mechanisms for this observation. The observed number of Foxp3+ cells in the peripheral blood was unchanging between SjS, RA, and healthy controls [106]. It is possible that the observations of increased numbers of Treg cells in the salivary glands of patients with SjS could be a result of proliferative enrichment due to increased costimulato‐ ry signaling (CD28 and CD86), and may have altered suppressive function as a result.

Costimulatory Molecules in Rheumatic Diseases Revisited with an Emphasis on Their Roles in…

http://dx.doi.org/10.5772/54480

109

In autoimmune diseases, dysfunction of Treg cells is a potential contributor to disease devel‐ opment, where costimulatory requirements for Treg cell proliferation and suppression capa‐ bilities may not be met. Based on reviewed research on autoimmune diseases, it appears that contribution of CD86 to Teff cells via CD28 signaling are potentially one of the initiating factors of disease, at least in the cases RA, autoimmune diabetes, and SjS. The lack of CD80 regulatory effects on these diseases appears in general to be due to a lack of expression on certain cell types, such as resident epithelium as in RA and SjS. In the previously mentioned model where CD80 helps suppressive Treg function, this relative lack of CD80 earlier in dis‐ ease onset could be contributing to the allowance of autoreactive T-cells to respond towards self-antigens. However, regulatory mechanisms of B7 ligand expression as well as the differ‐ ential signaling mechanisms that control Treg maintenance and function with regards to CD80 and CTLA-4 signaling pathways have yet to be elucidated. Some of the previously mentioned contradictory findings as to abnormal expression of B7 molecules and their func‐ tion in autoimmune diseases could be explained by multiple factors such as genetic back‐ ground, relative expression of B7 ligands, immune microenvironment, and timing of critical immunological events. Better understanding costimulatory and inhibitory requirements of CD80 and CD86 are worth further looking into since clinical trials involving CTLA-4-Ig (Abatacept, Orencia) have shown promise in clinical improvement for RA [107] but not so well in controlling flares in patients with non-life threatening SLE [108]. Clinical trials are also underway for MS and Type I diabetes, where findings do show efficacy in controlling disease activity in MS [109] and delaying beta cell loss in diabetes patients [110]. There is no data available for use of CTLA-4-Ig in SjS patients. However, the previously mentioned findings with CTLA-4-Ig in SjS-prone mice do show promise for the use of costimulatory blockade in prevention of disease [99]. Mechanisms involving these observed protective ef‐ fects of blocking B7 with regards to Treg maintenance and function should also be explored, since regulation of specific Treg populations may contribute to development of autoimmun‐ ity. Overall, there are still many questions as to how the CD86/CD80:CD28/CTLA-4 family interacts to form specific immune responses in vivo, especially in the areas of Treg develop‐

**4. Conclusions**

Results regarding Treg involvement in the pathogenesis of SjS have been contradictory. However, increasing evidence details defects in Treg contributing to disease. Recently it has also been shown that in situ patrolling Tregs are essential for protection against autoim‐ mune exocrinopathy. In this system, CCR7 deficient mice were unable to allow Treg egress from lymph nodes into peripheral tissues such as salivary and lacrimal glands, and that this resulted in disease resembling SjS [101]. Interestingly, wild-type C57BL/6 mice evaluation showed approximately 30% lacrimal and 23% salivary expression of Foxp3+ cells under steady-state conditions, while CCR7-deficient mice had approximately 7% and 9% respec‐ tively [101]. The Foxp3-deficient scurfy mouse model (essentially deficient in Treg cells) adds more complexity to the issue of roles of Tregs, since these mice do not develop inflam‐ mation or inflammatory cell infiltration into their salivary or lacrimal glands, however, adoptive transfer to Rag-deficient mice induces multi-organ inflammation in salivary, lacri‐ mal glands, stomach, small intestine, pancreas, colon, and even skeletal muscle. This includ‐ ed inflammation in the lacrimal and salivary glands as well as inhibited salivary function, where infiltration was located primarily in the acini and granular convoluted tubules [102]. SjS patients have also been shown to have decreased number of Tregs (CCR7+Foxp3+) in salivary glands compared to controls [101]. In line with this direct tissue evidence, there are also several other reports of fewer salivary Tregs present in patients with SjS [103, 104] and reports of Tregs with decreased suppression capabilities [105]. Reports of increased number of Foxp3+ Treg cells in the salivary glands of patients with SjS [105, 106], was positively cor‐ related with the grade of infiltration as evaluated by Chisholm score [106]. The authors do not address potential mechanisms for this observation. The observed number of Foxp3+ cells in the peripheral blood was unchanging between SjS, RA, and healthy controls [106]. It is possible that the observations of increased numbers of Treg cells in the salivary glands of patients with SjS could be a result of proliferative enrichment due to increased costimulato‐ ry signaling (CD28 and CD86), and may have altered suppressive function as a result.

#### **4. Conclusions**

human patients (data unpublished, [96]). Presumably, these cells are salivary gland epithe‐ lial cells, where the expression of low level costimulatory ligands is presumed normal and possibly protective. As for mouse models for SjS, retinoblastoma-associated protein 48 (RbAp48) transgenic mice, using a salivary gland specific promoter [97], resulted in the de‐ velopment of SjS-like symptoms. In salivary gland tissues of these mice the protein expres‐ sion of MHC class II, CD86, CD80, and ICAM-1 were all upregulated in affected mice salivary glands. Where the expression levels of CD86 is higher than CD80 in the SMX, indi‐ cating a potential pathogenic role of CD86 to disease initiation [96]. Another mouse model of SjS, the C57BL/6.NOD-*Aec1Aec2* is a double congenic mouse model of primary SjS syn‐ drome that contains two genetic regions (Idd3 and Idd5) derived from the NOD mouse model [98]. These genetic regions confer spontaneous development of SjS-like syndrome on the B6 background. A recent study using this model highlighted the importance of local B7 molecules and signaling through CD28 to disease progression [99]. In this report AAV trans‐ duced expression of the CTLA-4-Ig (blocks B7 molecules interactions) in the salivary glands of SjS-prone mouse model. Delivery of this gene construct via AAV prior to disease onset prevented glandular damage and lymphocytic cell infiltration commonly associated with disease, as well as, prevented loss of saliva secretion in the treated mice [99]. This was also accompanied by a significant increase in transforming growth factor beta-1 (TGF-B1) in the salivary glands and draining lymph nodes of these mice [99]. These results suggest a poten‐ tial regulatory role (either Treg or epithelial cells) involved with treatment of CTLA-4-Ig. Authors of this paper did not explore the mechanisms of action of this molecule. Along simi‐ lar lines, another study involving treatment of blocking CD86 MAbs to another mouse mod‐ el of primary SjS, the NFS/sld 3-day thymectomized mutant, was shown to prevent the autoimmune lesions and autoantibody production to a-fodrin [100]. Along with previous lines of research indicating a negative regulatory role of CD80, no significant effects were seen in mice treated with anti-CD80 MAbs [100]. These results together outline the impor‐ tance of costimulation to disease onset and severity and highlight the influence of CD86-

108 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

CD28 as a potential mediator of disease initiation in salivary and lacrimal glands.

Results regarding Treg involvement in the pathogenesis of SjS have been contradictory. However, increasing evidence details defects in Treg contributing to disease. Recently it has also been shown that in situ patrolling Tregs are essential for protection against autoim‐ mune exocrinopathy. In this system, CCR7 deficient mice were unable to allow Treg egress from lymph nodes into peripheral tissues such as salivary and lacrimal glands, and that this resulted in disease resembling SjS [101]. Interestingly, wild-type C57BL/6 mice evaluation showed approximately 30% lacrimal and 23% salivary expression of Foxp3+ cells under steady-state conditions, while CCR7-deficient mice had approximately 7% and 9% respec‐ tively [101]. The Foxp3-deficient scurfy mouse model (essentially deficient in Treg cells) adds more complexity to the issue of roles of Tregs, since these mice do not develop inflam‐ mation or inflammatory cell infiltration into their salivary or lacrimal glands, however, adoptive transfer to Rag-deficient mice induces multi-organ inflammation in salivary, lacri‐ mal glands, stomach, small intestine, pancreas, colon, and even skeletal muscle. This includ‐ ed inflammation in the lacrimal and salivary glands as well as inhibited salivary function, where infiltration was located primarily in the acini and granular convoluted tubules [102].

In autoimmune diseases, dysfunction of Treg cells is a potential contributor to disease devel‐ opment, where costimulatory requirements for Treg cell proliferation and suppression capa‐ bilities may not be met. Based on reviewed research on autoimmune diseases, it appears that contribution of CD86 to Teff cells via CD28 signaling are potentially one of the initiating factors of disease, at least in the cases RA, autoimmune diabetes, and SjS. The lack of CD80 regulatory effects on these diseases appears in general to be due to a lack of expression on certain cell types, such as resident epithelium as in RA and SjS. In the previously mentioned model where CD80 helps suppressive Treg function, this relative lack of CD80 earlier in dis‐ ease onset could be contributing to the allowance of autoreactive T-cells to respond towards self-antigens. However, regulatory mechanisms of B7 ligand expression as well as the differ‐ ential signaling mechanisms that control Treg maintenance and function with regards to CD80 and CTLA-4 signaling pathways have yet to be elucidated. Some of the previously mentioned contradictory findings as to abnormal expression of B7 molecules and their func‐ tion in autoimmune diseases could be explained by multiple factors such as genetic back‐ ground, relative expression of B7 ligands, immune microenvironment, and timing of critical immunological events. Better understanding costimulatory and inhibitory requirements of CD80 and CD86 are worth further looking into since clinical trials involving CTLA-4-Ig (Abatacept, Orencia) have shown promise in clinical improvement for RA [107] but not so well in controlling flares in patients with non-life threatening SLE [108]. Clinical trials are also underway for MS and Type I diabetes, where findings do show efficacy in controlling disease activity in MS [109] and delaying beta cell loss in diabetes patients [110]. There is no data available for use of CTLA-4-Ig in SjS patients. However, the previously mentioned findings with CTLA-4-Ig in SjS-prone mice do show promise for the use of costimulatory blockade in prevention of disease [99]. Mechanisms involving these observed protective ef‐ fects of blocking B7 with regards to Treg maintenance and function should also be explored, since regulation of specific Treg populations may contribute to development of autoimmun‐ ity. Overall, there are still many questions as to how the CD86/CD80:CD28/CTLA-4 family interacts to form specific immune responses in vivo, especially in the areas of Treg develop‐ ment, homeostasis, and suppression function. It is well established that Treg cells are impor‐ tant to the prevention of autoimmunity. Whether targeting costimulatory molecules to drive desirable Treg development and function to a complete reversal of disease phenotype needs to be further clarified in autoimmune diseases, especially in SjS.

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#### **Author details**

Adrienne E. Gauna and Seunghee Cha\*

Oral and Maxillofacial Diagnostic Sciences, University of Florida, Gainesville, USA

#### **References**

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ment, homeostasis, and suppression function. It is well established that Treg cells are impor‐ tant to the prevention of autoimmunity. Whether targeting costimulatory molecules to drive desirable Treg development and function to a complete reversal of disease phenotype needs

Oral and Maxillofacial Diagnostic Sciences, University of Florida, Gainesville, USA

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**Chapter 6**

**Immune Synapses Between Lymphocytes and Target**

The immune synapse is the interface between an antigen-presenting cell and a lymphocyte [1-4] as well as the interface between different lymphocytes, Natural Killer cells, and target cells [5]. This intercellular connection serves as a focal point for exocytosis and endocytosis [6]. Numerous investigations have elucidated the structure of the immunological synapse. The synapse is composed of a central region: a central supramolecular activation complex SMAC [cSMAC], a T cell receptor (TCR) cluster and associated signaling proteins, and peripheral SMAC ( pSMAC) of a ring of tight adhesion between the reacting cells [7]. The separated space of SMAC is the place of exocytic and endocytic events in this site but the precise site of signaling is not known [8]. The early signaling process occurs in peripheral microclusters in the pSMAC and the cSMAC in T and B cell synapses [9-12]. CD4 T cells form long-lived synapses with APCs – the synapses live few hours. CD8 T cells form transient synapses, lasting only minutes, because the target cells are killed [13,14]. In this cytotoxic synapse, activated Src kinases were detected in the cSMAC [13]. The cSMACs play an important role not only in signaling but also in receptor recycling because endosomal compartments polarize to the point immediately beneath the cSMAC of the immunological synapse [15]. The endosome comes to lie underneath the cSMAC as polarization of the microtubule skeletons occurs during synapse formation. This polarization is antigen-dependent. The receptor activation leads to accumulation of actin across the synapse and formation of an outer ring around the synapse [16,17]. The cytotoxic reaction of lymphocytes CD8 is connected with release of specialized lysosomes containing the lytic pore-forming protein perforin, which enables gransymes to lead to rapid apoptosis of the target cell [17]; the centrosome in the lymphocytes is polarized right up to the plasma membrane containing the synapse cSMAC [18]. The lytic granules are delivered to a specialized secretory domain within the synapse by moving along the microtubules toward the centro‐

> © 2013 Ben-Skowronek and Ciechanek; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is

> © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

and reproduction in any medium, provided the original work is properly cited.

**Cells in Autoimmune Thyroid Diseases**

Iwona Ben-Skowronek and Roman Ciechanek

Additional information is available at the end of the chapter

properly cited.

http://dx.doi.org/10.5772/53009

**1. Introduction**

### **Immune Synapses Between Lymphocytes and Target Cells in Autoimmune Thyroid Diseases**

Iwona Ben-Skowronek and Roman Ciechanek

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53009

#### **1. Introduction**

The immune synapse is the interface between an antigen-presenting cell and a lymphocyte [1-4] as well as the interface between different lymphocytes, Natural Killer cells, and target cells [5]. This intercellular connection serves as a focal point for exocytosis and endocytosis [6]. Numerous investigations have elucidated the structure of the immunological synapse. The synapse is composed of a central region: a central supramolecular activation complex SMAC [cSMAC], a T cell receptor (TCR) cluster and associated signaling proteins, and peripheral SMAC ( pSMAC) of a ring of tight adhesion between the reacting cells [7]. The separated space of SMAC is the place of exocytic and endocytic events in this site but the precise site of signaling is not known [8]. The early signaling process occurs in peripheral microclusters in the pSMAC and the cSMAC in T and B cell synapses [9-12]. CD4 T cells form long-lived synapses with APCs – the synapses live few hours. CD8 T cells form transient synapses, lasting only minutes, because the target cells are killed [13,14]. In this cytotoxic synapse, activated Src kinases were detected in the cSMAC [13]. The cSMACs play an important role not only in signaling but also in receptor recycling because endosomal compartments polarize to the point immediately beneath the cSMAC of the immunological synapse [15]. The endosome comes to lie underneath the cSMAC as polarization of the microtubule skeletons occurs during synapse formation. This polarization is antigen-dependent. The receptor activation leads to accumulation of actin across the synapse and formation of an outer ring around the synapse [16,17]. The cytotoxic reaction of lymphocytes CD8 is connected with release of specialized lysosomes containing the lytic pore-forming protein perforin, which enables gransymes to lead to rapid apoptosis of the target cell [17]; the centrosome in the lymphocytes is polarized right up to the plasma membrane containing the synapse cSMAC [18]. The lytic granules are delivered to a specialized secretory domain within the synapse by moving along the microtubules toward the centro‐

properly cited.

© 2013 Ben-Skowronek and Ciechanek; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

some. Granule contents are then released into a small cleft between the two cells [17]. Since the overall levels of surface and endocytosed proteins remain the same regardless of ICAM-1, this suggests that ICAM-1–LFA-1 engagement in the pSMAC acts to restrict and focus endocytic and exocytic events to the center of the synapse. Griffiths suggests that the centro‐ some may play a role in identifying a specialized area of membrane for focal endocytosis and exocytosis [6]. An important role in formation of the immunological synapse is played by the localization of mitochondria – the mitochondria can activate and terminate the activity of immune synapses [19]. The *in vivo* image of T cell activation is slightly more complex. In an experimental system that uses subcutaneous injection of labeled LPS-activated dendritic cells followed by intravenous injection of naive transgenic CD8+ T cells, behavior of these cells and a three-phase model for T cell activation were observed: [20]. Phase 1 includes initial transient T cell–DC interactions characterized by continued rapid T cell migration that can last from 30 min to 8 h depending on the pMHC density. Signals in phase 1 are integrated through kinapses. Phase 2 is a period of stable T cell–DC interactions lasting ∼12 h, during which cytokines such as IL-2 are produced. Signals in phase 2 are integrated through the immune synapse. Phase 3 is a return to transient T cell–DC interaction and rapid T cell migration during which the T cell divides multiple times and then exits the lymphoid tissue. The correct interpretation of these stop and go signals is critical for generation of effector and memory T cells [21,22].

standard pathologic investigations. All the children were in euthyreosis [Tab.1]. All children's

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All the patients received physical examination to assess the goiter and clinical signs and symptoms of thyroid disorders. The TSH (Thyroid-stimulating hormone), fT4 (free thyroxin) and TT3 (total triiodothyronine) hormones were assayed by MEIA (Abbott Kit, Langford, Ireland). The levels of TSH receptor antibodies were measured by RIA (TRAK assay BRAHMS Diagnostica GmbH, Berlin, Germany). The thyroperoxidase (TPO) and thyroglobulin (TG) antibodies were assayed by LIA (Lumitest BRAHMS Diagnostica GmbH, Berlin, Germany).

In the patients with Graves' disease, symptoms of thyrotoxicosis: goiter, tachycardia, sleep‐ lessness, anxiety, high diastolic/systolic blood pressure amplitude, an increase in fT4 (mean 3.8± 0.7 ng/dl) and TT3 (mean 363±175.3 ng/dl), and a decrease in TSH (mean 0.004±0.003 mU/ l) were observed. The levels of antibodies against the TSH receptor (TRAB) (7-462U/ml) and the levels of TPO antibodies (21-6663U/ml) and TG antibodies (25-13351U/ml) were usually increased. The patients were treated with methimazole in initial doses 0.9-0.5 mg/kg b.w./day during 4-6 weeks and after that time, when in euthyreosis, they got maintenance doses c.a.0.1 mg/kg b.w./ day (mainly 5mg/day) in combination with a low dose of l-thyroxin (25μg/day) during 18-24 months. Children with Graves' disease, whose early relapses of hyperthyreosis necessitated surgery treatment – thyroidectomy after 18-36 months, were qualified for the

Hashimoto's thyroiditis was recognized in patients with parenchymal or nodular large size goiter accompanied by pressure to other neck structures in the phase of euthyreosis or hypothyreosis, rarely in hyperthyreosis (Hashitoxicosis). In ultrasonography, a non-homoge‐ nous structure of the thyroid was observed. The levels of TPO Ab and TG Ab were increased, but the levels of the TRAb were in normal ranges. In histopathological examination, mono‐ nuclear lymphatic infiltrations in the thyroid parenchyma were detected, and Hashimoto's thyroiditis was diagnosed. Before surgery, the patients were usually treated with l-thyroxin

> **TSH mIU/L**

Graves' disease 30 5-19 0,001-0,005 3,3-5,1 21-6663 25-13351 7-462

Control group 30 6-19 0,270-4,200 x x x x Normal ranges 0,270-4,200 0,8-2,3 <34 <115 <1

**fT4 ng/dl**

30 8-19 0,600-98,800 0,1—2,3 132-9856 128-14567 0-0,99

**TPO Ab IU/L**

**TG Ab IU/L**

**TSI IU/L**

parents signed an informed consent before autopsy or surgical operation.

All patients' parents signed an informed consent before these investigations.

*2.1.2. Patient qualification procedure*

investigation [Tab. 1].

25-100 μg/day. [Tab. 1].

Hashimoto's thyroiditis

**Patients number**

**Table 1.** Characteristics of the patients examined before treatment

**Age [years]**

**The aim** of the study is presentation of the ultrastructure of immune synapses between T- cells and plasma cells and target cells *in vivo* in autoimmune thyroid diseases.

#### **2. Material and methods**

#### **2.1. Patients**

A group of children and adolescents was chosen for the study to exclude the impact of aging processes and other diseases connected with age: circulatory disorders, arterial sclerosis, and drug use.

The study involved 90 children: 30 children affected with Graves' disease, 30 children with Hashimoto's thyroiditis and 30 children as a control group. The children were treated in the Department of Pediatric Endocrinology and Neurology in Lublin and in the Pediatric Depart‐ ment in Rzeszow in the years 1994 – 2007 and operated on in the Surgery Department of the Regional Hospital in Lublin and in the Regional Hospital in Rzeszow.

The investigation was accepted by the local Ethical Committee at the Medical University in Lublin.

#### *2.1.1. Control group*

The control group consisted of 30 children aged 6-19 who had died in accidents and of other non-autoimmune diseases; the thyroid specimens were taken during autopsy (n=25). Some specimens were taken during a surgical operation of thyroglossal cysts and during surgery of parathyroid glands (n=5). These were fragments of routinely sampled tissue specimens for standard pathologic investigations. All the children were in euthyreosis [Tab.1]. All children's parents signed an informed consent before autopsy or surgical operation.

#### *2.1.2. Patient qualification procedure*

some. Granule contents are then released into a small cleft between the two cells [17]. Since the overall levels of surface and endocytosed proteins remain the same regardless of ICAM-1, this suggests that ICAM-1–LFA-1 engagement in the pSMAC acts to restrict and focus endocytic and exocytic events to the center of the synapse. Griffiths suggests that the centro‐ some may play a role in identifying a specialized area of membrane for focal endocytosis and exocytosis [6]. An important role in formation of the immunological synapse is played by the localization of mitochondria – the mitochondria can activate and terminate the activity of immune synapses [19]. The *in vivo* image of T cell activation is slightly more complex. In an experimental system that uses subcutaneous injection of labeled LPS-activated dendritic cells followed by intravenous injection of naive transgenic CD8+ T cells, behavior of these cells and a three-phase model for T cell activation were observed: [20]. Phase 1 includes initial transient T cell–DC interactions characterized by continued rapid T cell migration that can last from 30 min to 8 h depending on the pMHC density. Signals in phase 1 are integrated through kinapses. Phase 2 is a period of stable T cell–DC interactions lasting ∼12 h, during which cytokines such as IL-2 are produced. Signals in phase 2 are integrated through the immune synapse. Phase 3 is a return to transient T cell–DC interaction and rapid T cell migration during which the T cell divides multiple times and then exits the lymphoid tissue. The correct interpretation of these

120 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

stop and go signals is critical for generation of effector and memory T cells [21,22].

and plasma cells and target cells *in vivo* in autoimmune thyroid diseases.

Regional Hospital in Lublin and in the Regional Hospital in Rzeszow.

**2. Material and methods**

**2.1. Patients**

drug use.

Lublin.

*2.1.1. Control group*

**The aim** of the study is presentation of the ultrastructure of immune synapses between T- cells

A group of children and adolescents was chosen for the study to exclude the impact of aging processes and other diseases connected with age: circulatory disorders, arterial sclerosis, and

The study involved 90 children: 30 children affected with Graves' disease, 30 children with Hashimoto's thyroiditis and 30 children as a control group. The children were treated in the Department of Pediatric Endocrinology and Neurology in Lublin and in the Pediatric Depart‐ ment in Rzeszow in the years 1994 – 2007 and operated on in the Surgery Department of the

The investigation was accepted by the local Ethical Committee at the Medical University in

The control group consisted of 30 children aged 6-19 who had died in accidents and of other non-autoimmune diseases; the thyroid specimens were taken during autopsy (n=25). Some specimens were taken during a surgical operation of thyroglossal cysts and during surgery of parathyroid glands (n=5). These were fragments of routinely sampled tissue specimens for All patients' parents signed an informed consent before these investigations.

All the patients received physical examination to assess the goiter and clinical signs and symptoms of thyroid disorders. The TSH (Thyroid-stimulating hormone), fT4 (free thyroxin) and TT3 (total triiodothyronine) hormones were assayed by MEIA (Abbott Kit, Langford, Ireland). The levels of TSH receptor antibodies were measured by RIA (TRAK assay BRAHMS Diagnostica GmbH, Berlin, Germany). The thyroperoxidase (TPO) and thyroglobulin (TG) antibodies were assayed by LIA (Lumitest BRAHMS Diagnostica GmbH, Berlin, Germany).

In the patients with Graves' disease, symptoms of thyrotoxicosis: goiter, tachycardia, sleep‐ lessness, anxiety, high diastolic/systolic blood pressure amplitude, an increase in fT4 (mean 3.8± 0.7 ng/dl) and TT3 (mean 363±175.3 ng/dl), and a decrease in TSH (mean 0.004±0.003 mU/ l) were observed. The levels of antibodies against the TSH receptor (TRAB) (7-462U/ml) and the levels of TPO antibodies (21-6663U/ml) and TG antibodies (25-13351U/ml) were usually increased. The patients were treated with methimazole in initial doses 0.9-0.5 mg/kg b.w./day during 4-6 weeks and after that time, when in euthyreosis, they got maintenance doses c.a.0.1 mg/kg b.w./ day (mainly 5mg/day) in combination with a low dose of l-thyroxin (25μg/day) during 18-24 months. Children with Graves' disease, whose early relapses of hyperthyreosis necessitated surgery treatment – thyroidectomy after 18-36 months, were qualified for the investigation [Tab. 1].

Hashimoto's thyroiditis was recognized in patients with parenchymal or nodular large size goiter accompanied by pressure to other neck structures in the phase of euthyreosis or hypothyreosis, rarely in hyperthyreosis (Hashitoxicosis). In ultrasonography, a non-homoge‐ nous structure of the thyroid was observed. The levels of TPO Ab and TG Ab were increased, but the levels of the TRAb were in normal ranges. In histopathological examination, mono‐ nuclear lymphatic infiltrations in the thyroid parenchyma were detected, and Hashimoto's thyroiditis was diagnosed. Before surgery, the patients were usually treated with l-thyroxin 25-100 μg/day. [Tab. 1].

**Table 1.** Characteristics of the patients examined before treatment

#### **3. Ultrastructural investigations**

Specimens for ultrastructural investigations were obtained during thyroidectomy. Small segments of thyroid were cut into 0,5mm³ pieces and fixed in 4% glutaraldehyde in 0,1 M cacodylate buffer, pH 7.4 for 24 h in 4ºC, post fixed in 2% OsO4 in the same buffer for 1h in room temperature, dehydrated in a graded series (up to 100%) of ethanol and embedded in 812 Epon. They were then polymerized at 60ºC. Five specimens were taken from every thyroid from each patient with Graves' disease, Hashimoto's thyroiditis, and from the control group. Epon blocks were cut with an RMC MT-7 ultramicrotome, USA. From every specimen were analyzed serial 10 slides. Ultrathin sections were contrasted with uranyl acetate and lead citrate and examined under the EM 900 Zeiss Germany Electron Microscope.

#### **4. Results**

In the control group of children without a thyroid disease, lymphocytes in the interstitium of the thyroid gland were observed sporadically. The lymphatic cells did not cross the basal membrane of thyroid follicles, were not in contact with thyrocytes and did not form groups. In Graves' disease, T cells that crossed the basal membrane of the vesicles were observed to be in contact with thyrocytes. The lymphocytes migrated to thyroid follicles from capillary vessels or from lymphatic follicles. The migrating T-cells had numerous projections – lamel‐ lipodia on their surface. Polarization of cell organelles was already visible in narrow capillaries. The lamellipodia, mitochondria, and the Golgi system were located in the same part of the lymphocyte [Fig.1]. T cells, which penetrated across the basal membrane between thyrocytes, looked similar [Fig. 2]. The T- cells formed numerous junctions with thyrocytes. The structure of these connections was similar to zonula occludens with an area of cell membrane fusion and area of free spaces between cells, in which protein substances were secreted [Fig.2]. The T-cells were not polarized in those connections with thyrocytes. The thyrocytes were not damaged, but were active and had numerous mitochondria, secretory vesicles and a big amount of euchromatin in the nuclei [Fig. 2].

In Hashimoto's thyroiditis, the sites of contact between T-cells and lymphocytes had the character of an immune synapse, too. The synapse, however, looked different. The T-cells were polarized – the centrioles, mitochondria, Golgi system, and secretory vesicles were present in the part connected with the thyrocytes. The synapse was composed of a distal part – an adhesion zone, and a central part – a space in which electron dense substances were secreted [Fig.3]. The thyrocytes staying in contact with T-cells exhibited the features of apoptosis: dark, concentrated heterochromatin in the nucleus, swollen mitochondria, and enlarged cisterns of endoplasmic reticulum.

In AITD, synapses between plasma cells and thyrocytes were observed. In Graves' disease, synapses were formed in the distal part – zonula adherens - without fusion of thyrocyte and plasmocyte cell membranes and in the central part - the space between membrane of plasma cells and thyrocytes. Electron dense substances from the rough endoplasmic reticulum of the plasma cells – most probably immunoglobulins - were secreted to this space [Fig.4]. Immu‐ noglobulins encrusted the basal membrane of thyrocytes.The thyroid cells staying in contact with plasma cells were active: with a big amount of euchromatin in the nucleus, numerous

**Figure 2.** The lymphocyte between thyrocytes in the thyroid follicle in Graves' disease. The lymphocyte formed numer‐ ous immune synapses with the thyrocyte. The immune synapses are limited by zonula occludens (pSMAC with fusion of cell membranes). The space (cSMAC) is visible in the center of the immune synapse. The thyrocytes are active with‐ out signs of damage. RER – rough endoplasmic reticulum, M-mitochondria, V-secretory vesicle, MB- basal membrane,

**Figure 1.** The lymphocyte migrating in the capillary vessel. Polarization of the lymphatic cell is visible: lamellipodia, mitochondria, and Golgi complex are present on the same side. N- nucleus, M- mitochondria, G-Golgi system, RBC- red

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secretory vesicles, and abundant microvilli [Fig.4].

IS- immune synapse. TEM magn. 15 000x.

blood cell. TEM magn. 15 000x

**3. Ultrastructural investigations**

122 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

**4. Results**

euchromatin in the nuclei [Fig. 2].

endoplasmic reticulum.

Specimens for ultrastructural investigations were obtained during thyroidectomy. Small segments of thyroid were cut into 0,5mm³ pieces and fixed in 4% glutaraldehyde in 0,1 M cacodylate buffer, pH 7.4 for 24 h in 4ºC, post fixed in 2% OsO4 in the same buffer for 1h in room temperature, dehydrated in a graded series (up to 100%) of ethanol and embedded in 812 Epon. They were then polymerized at 60ºC. Five specimens were taken from every thyroid from each patient with Graves' disease, Hashimoto's thyroiditis, and from the control group. Epon blocks were cut with an RMC MT-7 ultramicrotome, USA. From every specimen were analyzed serial 10 slides. Ultrathin sections were contrasted with uranyl acetate and lead citrate

In the control group of children without a thyroid disease, lymphocytes in the interstitium of the thyroid gland were observed sporadically. The lymphatic cells did not cross the basal membrane of thyroid follicles, were not in contact with thyrocytes and did not form groups. In Graves' disease, T cells that crossed the basal membrane of the vesicles were observed to be in contact with thyrocytes. The lymphocytes migrated to thyroid follicles from capillary vessels or from lymphatic follicles. The migrating T-cells had numerous projections – lamel‐ lipodia on their surface. Polarization of cell organelles was already visible in narrow capillaries. The lamellipodia, mitochondria, and the Golgi system were located in the same part of the lymphocyte [Fig.1]. T cells, which penetrated across the basal membrane between thyrocytes, looked similar [Fig. 2]. The T- cells formed numerous junctions with thyrocytes. The structure of these connections was similar to zonula occludens with an area of cell membrane fusion and area of free spaces between cells, in which protein substances were secreted [Fig.2]. The T-cells were not polarized in those connections with thyrocytes. The thyrocytes were not damaged, but were active and had numerous mitochondria, secretory vesicles and a big amount of

In Hashimoto's thyroiditis, the sites of contact between T-cells and lymphocytes had the character of an immune synapse, too. The synapse, however, looked different. The T-cells were polarized – the centrioles, mitochondria, Golgi system, and secretory vesicles were present in the part connected with the thyrocytes. The synapse was composed of a distal part – an adhesion zone, and a central part – a space in which electron dense substances were secreted [Fig.3]. The thyrocytes staying in contact with T-cells exhibited the features of apoptosis: dark, concentrated heterochromatin in the nucleus, swollen mitochondria, and enlarged cisterns of

In AITD, synapses between plasma cells and thyrocytes were observed. In Graves' disease, synapses were formed in the distal part – zonula adherens - without fusion of thyrocyte and plasmocyte cell membranes and in the central part - the space between membrane of plasma cells and thyrocytes. Electron dense substances from the rough endoplasmic reticulum of the

and examined under the EM 900 Zeiss Germany Electron Microscope.

**Figure 1.** The lymphocyte migrating in the capillary vessel. Polarization of the lymphatic cell is visible: lamellipodia, mitochondria, and Golgi complex are present on the same side. N- nucleus, M- mitochondria, G-Golgi system, RBC- red blood cell. TEM magn. 15 000x

**Figure 2.** The lymphocyte between thyrocytes in the thyroid follicle in Graves' disease. The lymphocyte formed numer‐ ous immune synapses with the thyrocyte. The immune synapses are limited by zonula occludens (pSMAC with fusion of cell membranes). The space (cSMAC) is visible in the center of the immune synapse. The thyrocytes are active with‐ out signs of damage. RER – rough endoplasmic reticulum, M-mitochondria, V-secretory vesicle, MB- basal membrane, IS- immune synapse. TEM magn. 15 000x.

plasma cells – most probably immunoglobulins - were secreted to this space [Fig.4]. Immu‐ noglobulins encrusted the basal membrane of thyrocytes.The thyroid cells staying in contact with plasma cells were active: with a big amount of euchromatin in the nucleus, numerous secretory vesicles, and abundant microvilli [Fig.4].

In Hashimoto's thyroiditis, polarization of plasma cells was observed; the centrioles and Golgi system, mitochondria and well-developed rough endoplasmic reticulum were observed in the part connected with thyrocytes. In some areas of the contact places, secretion of substances with medium electron density from plasma cells to thyrocytes was observed [Fig.5]. The plasma cells adhered in a large area to thyrocytes, and the thyrocytes exhibited features of damage and destruction: fragmentation of the endoplasmic reticulum, edema of mitochondria, and condensation of the chromatin in the nucleus. In advanced stages, destruction and

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**Figure 5.** The plasma cell in contact with thyrocyte in Hashimoto's thyroiditis. The immune synapse is composed of a surrounding adhesion zone (pSMAC) in the central part of the site of exocytosis of electron dense substances and vesi‐ cles from plasma cell. Polarization of the organelle is visible in the plasma cell: centrioles, lysosomes, and mitochondria in the region of immune synapse. The thyrocyte is damaged with edema of mitochondria and destruction of endo‐ plasmic reticulum. C-centriole, M-mitochondrium, RER- rough endoplasmic reticulum, N-nucleus. TEM magm.25 000x

The immune synapses occurring in the thyroids of patients with Graves' disease were similar to the synapse described by Dustin [23]. Dynamic studies with planar bilayers further showed that the immune synapse was formed through a nascent intermediate in which activating TCR clusters are formed first in the pSMAC and then move to the cSMAC region in an F-actin-

In the connection between thyrocytes and T-cells, the zonula occludens in Graves' disease and zonula adherens in Hashimoto's thyroiditis seem to be the peripheral pSMAC and the space in the center can correspond to the central supramolecular activation complex cSMAC.

dependent process in a few minutes to form the pattern [7].

fragmentation of thyrocytes were observed.

**5. Discussion**

**Figure 3.** The lymphocyte between thyroid follicles in Hashimoto's thyroiditis. The lymphocyte is connected with the thyrocyte by an immune synapse limited by zonula adherens without cell membrane fusion (pSMAC). The space (cSMAC) is visible in the center of the immune synapse. The lymphocyte organelles are polarized under the synapse: centriole, mitochondria, granules, and Golgi complex. Signs of damage are present in the thyrocyte: enlarged cisterns of rough endoplasmic reticulum. C-centriole, M-mitochondria, G-Golgi system, L-lysosome, RER- rough endoplasmic reticulum, N-nucleus. TEM magm.25 000x

**Figure 4.** The plasma cells in contact with thyrocytes in Graves' disease. a) The space of the immune synapse in which electron dense substances, probably antibodies, are secreted. Enlarged cisterns of endoplasmic reticulum were ob‐ served in the thyrocyte. RER-rough endoplasmic reticulum, CM cell membrane, \* immunoglobulins. TEM magn. 25 000x. b)The advanced phase of the immune reaction: the deposits of immunoglobulins in the space between the thy‐ rocyte and plasmocyte. The space of the immune synapse is limited by zonula adherens. CM – cell membrane, RER – rough endoplasmic reticulum, \* immunoglobulins deposit. TEM magn. 25 000x

In Hashimoto's thyroiditis, polarization of plasma cells was observed; the centrioles and Golgi system, mitochondria and well-developed rough endoplasmic reticulum were observed in the part connected with thyrocytes. In some areas of the contact places, secretion of substances with medium electron density from plasma cells to thyrocytes was observed [Fig.5]. The plasma cells adhered in a large area to thyrocytes, and the thyrocytes exhibited features of damage and destruction: fragmentation of the endoplasmic reticulum, edema of mitochondria, and condensation of the chromatin in the nucleus. In advanced stages, destruction and fragmentation of thyrocytes were observed.

**Figure 5.** The plasma cell in contact with thyrocyte in Hashimoto's thyroiditis. The immune synapse is composed of a surrounding adhesion zone (pSMAC) in the central part of the site of exocytosis of electron dense substances and vesi‐ cles from plasma cell. Polarization of the organelle is visible in the plasma cell: centrioles, lysosomes, and mitochondria in the region of immune synapse. The thyrocyte is damaged with edema of mitochondria and destruction of endo‐ plasmic reticulum. C-centriole, M-mitochondrium, RER- rough endoplasmic reticulum, N-nucleus. TEM magm.25 000x

#### **5. Discussion**

**Figure 4.** The plasma cells in contact with thyrocytes in Graves' disease. a) The space of the immune synapse in which electron dense substances, probably antibodies, are secreted. Enlarged cisterns of endoplasmic reticulum were ob‐ served in the thyrocyte. RER-rough endoplasmic reticulum, CM cell membrane, \* immunoglobulins. TEM magn. 25 000x. b)The advanced phase of the immune reaction: the deposits of immunoglobulins in the space between the thy‐ rocyte and plasmocyte. The space of the immune synapse is limited by zonula adherens. CM – cell membrane, RER –

**Figure 3.** The lymphocyte between thyroid follicles in Hashimoto's thyroiditis. The lymphocyte is connected with the thyrocyte by an immune synapse limited by zonula adherens without cell membrane fusion (pSMAC). The space (cSMAC) is visible in the center of the immune synapse. The lymphocyte organelles are polarized under the synapse: centriole, mitochondria, granules, and Golgi complex. Signs of damage are present in the thyrocyte: enlarged cisterns of rough endoplasmic reticulum. C-centriole, M-mitochondria, G-Golgi system, L-lysosome, RER- rough endoplasmic

rough endoplasmic reticulum, \* immunoglobulins deposit. TEM magn. 25 000x

reticulum, N-nucleus. TEM magm.25 000x

124 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

The immune synapses occurring in the thyroids of patients with Graves' disease were similar to the synapse described by Dustin [23]. Dynamic studies with planar bilayers further showed that the immune synapse was formed through a nascent intermediate in which activating TCR clusters are formed first in the pSMAC and then move to the cSMAC region in an F-actindependent process in a few minutes to form the pattern [7].

In the connection between thyrocytes and T-cells, the zonula occludens in Graves' disease and zonula adherens in Hashimoto's thyroiditis seem to be the peripheral pSMAC and the space in the center can correspond to the central supramolecular activation complex cSMAC.

An interesting observation is the difference in polarization of lymphocytes in immune synapses. The lymphocyte stimulating thyrocytes in Graves' disease were not polarized, but the cytotoxic lymphocytes in Hashimoto's thyroiditis had polarized organelles in the cytoplasm.

immunoglobulins deposits [35, 36] were observed. In Hashimoto's thyroiditis, the plasma cells were polarized and formed the microtubule organizing center (MTOC) consisting of centrioles, mitochondria and Golgi apparatus and probably microtubules and microfilaments. The peripheral zonula adherens surrounded the place of immunoglobulin secretion, but the immunoglobulins penetrated to thyrocytes and probably led to damage to these cells [38].

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**•** Immune synapses between T-cells and plasma cells with thyroid's epithelial cells were

**•** In the ultrastructure of the synapse, peripheral zonula occludens or zonula adherens and a

**•** The lymphocytes forming the cytotoxic synapse were characterized by presence of a

This work was supported by Grant 2P05E04327 from the Polish Ministry of Science and Higher

1 Department of Paediatric Endocrinology and Diabetology, Medical University in Lublin,,

**Ethics.** The investigation was accepted by the local Ethical Committee at the Medical Uni‐ versity in Lublin. All children's parents signed an informed consent before autopsy or surgi‐

central space were observed in all types of the immune synapses.

and Roman Ciechanek2

2 Division of Surgery, Provincial Specialist Hospital in Lublin,, Poland

**Competing interests.** The authors declare that they have no competing interests.

\*Address all correspondence to<EMAIL_ADDRESS>
\*Address all correspondence to<EMAIL_ADDRESS>
**6. Conclusions**

found in AITD.

microtubule-organizing center.

**Acknowledgements**

Education.

Poland

cal operation.

**Author details**

Iwona Ben-Skowronek1

The polarization of the T-cell, i.e. formation of a center with a centriole, mitochondria, and Golgi complex suggested special organization of the cellular tubules and filaments. Actin filaments (F-actin) play a critical role throughout the various stages of T cell activation. In the steady state, actin polymerization at the leading edge and cytoskeletal contraction at the lamelliopodium mediate rapid migration [24].The microtubule organizing center (MTOC) and microtubule network of the cell provide a molecular way for vesicle movement and structural support for polarized cell functions. Within seconds after TCR stimulation, the MTOC mobilizes and polarizes to the immune synapse in T cells. Polarization is important for efficient trafficking and directed secretion of cytolytic granules and cytokines for secretion at the synapse [25-27]

Previous studies [28-30] report that mitochondria accumulate at the immunological synapse following T-cell stimulation. The fusion factor DRP1 (dynamin-related protein 1) regulates mitochondria positioning close to the peripheral supramolecular activation cluster (pSMAC), which together with the central SMAC forms the immune synapse in T cells [19]. The immune synapse controls calcium signals and calcium-dependent T-cell functions [31]. Our observa‐ tions *in vivo* are similar to pictures from the electron microscope from investigations in cell culture published by Tsun [32].

Probably, the polarization of organelles in the cytotoxic lymphocytes observed is connected with transport of cytotoxic substances from these cells to thyrocytes.

Stinchcombe [33] observed NK cells conjugated with B-cells with glycolipid-pulsed CD1 bearing targets. High-resolution electron micrographs of the immunological synapse formed between NK and iNKT cytolytic cells with their targets revealed that, in both NK and iNKT cells, the centrioles could be found associated (or 'docked') with the plasma membrane within the immunological synapse. Secretory clefts were visible within the synapses formed by both NK and iNKT cells, and secretory lysosomes were polarized along microtubules leading towards the docked centrosome. The Golgi apparatus and recycling endosomes were also polarized towards the centrosome at the plasma membrane within the synapse [33]. It seems that the polarization process is connected with the cytotoxic interactions between T-cells and thyrocytes in Hashimoto's thyroiditis.

The immune synapse between plasma cells and APCs has been seldom described. Batista described the immunological synapse between plasma cells and antigen presenting cells [34]. We observed two types of immune synapses between plasma cells and thyrocytes in AITD. In Graves' disease, they are the stimulating synapses: immunoglobulins encrusting the basal membrane of the thyrocyte were secreted in the central space of the synapse and were probably connected with TSH- receptors. In the last phase of this process, deposits of immunoglobulins were visible. The similar change were observed in kidney [35,36]. In glomerulonephritis, subendothelial complement deposits [36,37] and sun epithelial (similar to situation in thyroid) immunoglobulins deposits [35, 36] were observed. In Hashimoto's thyroiditis, the plasma cells were polarized and formed the microtubule organizing center (MTOC) consisting of centrioles, mitochondria and Golgi apparatus and probably microtubules and microfilaments. The peripheral zonula adherens surrounded the place of immunoglobulin secretion, but the immunoglobulins penetrated to thyrocytes and probably led to damage to these cells [38].

#### **6. Conclusions**

An interesting observation is the difference in polarization of lymphocytes in immune synapses. The lymphocyte stimulating thyrocytes in Graves' disease were not polarized, but the cytotoxic lymphocytes in Hashimoto's thyroiditis had polarized organelles in the

126 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

The polarization of the T-cell, i.e. formation of a center with a centriole, mitochondria, and Golgi complex suggested special organization of the cellular tubules and filaments. Actin filaments (F-actin) play a critical role throughout the various stages of T cell activation. In the steady state, actin polymerization at the leading edge and cytoskeletal contraction at the lamelliopodium mediate rapid migration [24].The microtubule organizing center (MTOC) and microtubule network of the cell provide a molecular way for vesicle movement and structural support for polarized cell functions. Within seconds after TCR stimulation, the MTOC mobilizes and polarizes to the immune synapse in T cells. Polarization is important for efficient trafficking and directed secretion of cytolytic granules and cytokines for secretion at the

Previous studies [28-30] report that mitochondria accumulate at the immunological synapse following T-cell stimulation. The fusion factor DRP1 (dynamin-related protein 1) regulates mitochondria positioning close to the peripheral supramolecular activation cluster (pSMAC), which together with the central SMAC forms the immune synapse in T cells [19]. The immune synapse controls calcium signals and calcium-dependent T-cell functions [31]. Our observa‐ tions *in vivo* are similar to pictures from the electron microscope from investigations in cell

Probably, the polarization of organelles in the cytotoxic lymphocytes observed is connected

Stinchcombe [33] observed NK cells conjugated with B-cells with glycolipid-pulsed CD1 bearing targets. High-resolution electron micrographs of the immunological synapse formed between NK and iNKT cytolytic cells with their targets revealed that, in both NK and iNKT cells, the centrioles could be found associated (or 'docked') with the plasma membrane within the immunological synapse. Secretory clefts were visible within the synapses formed by both NK and iNKT cells, and secretory lysosomes were polarized along microtubules leading towards the docked centrosome. The Golgi apparatus and recycling endosomes were also polarized towards the centrosome at the plasma membrane within the synapse [33]. It seems that the polarization process is connected with the cytotoxic interactions between T-cells and

The immune synapse between plasma cells and APCs has been seldom described. Batista described the immunological synapse between plasma cells and antigen presenting cells [34]. We observed two types of immune synapses between plasma cells and thyrocytes in AITD. In Graves' disease, they are the stimulating synapses: immunoglobulins encrusting the basal membrane of the thyrocyte were secreted in the central space of the synapse and were probably connected with TSH- receptors. In the last phase of this process, deposits of immunoglobulins were visible. The similar change were observed in kidney [35,36]. In glomerulonephritis, subendothelial complement deposits [36,37] and sun epithelial (similar to situation in thyroid)

with transport of cytotoxic substances from these cells to thyrocytes.

cytoplasm.

synapse [25-27]

culture published by Tsun [32].

thyrocytes in Hashimoto's thyroiditis.

#### **Acknowledgements**

This work was supported by Grant 2P05E04327 from the Polish Ministry of Science and Higher Education.

#### **Author details**

Iwona Ben-Skowronek1 and Roman Ciechanek2

\*Address all correspondence to<EMAIL_ADDRESS>
\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Paediatric Endocrinology and Diabetology, Medical University in Lublin,, Poland

2 Division of Surgery, Provincial Specialist Hospital in Lublin,, Poland

**Competing interests.** The authors declare that they have no competing interests.

**Ethics.** The investigation was accepted by the local Ethical Committee at the Medical Uni‐ versity in Lublin. All children's parents signed an informed consent before autopsy or surgi‐ cal operation.

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**Section 2**

**Infectious Agents and Host Inflammatory**

**Response**

**Infectious Agents and Host Inflammatory Response**

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130 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

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**Chapter 7**

**Microbiome and Autoimmunity**

Natalie Cherepahina, Zaur Shogenov,

Jamilyia Tabaksoeva, Mikhail Paltsev and

Additional information is available at the end of the chapter

Most of the currently known infections represent a persistent and stepwise development of the canonical chronic diseases of infectious origin (CDIO) associated with conditionally pathogenic microorganisms, i.e., viruses, intracellular parasites and other representatives of the endogenous microbiome, particularly, those manifesting with atypical properties. Pathogens are able to interact between themselves and to cause a disease, and the patho‐ gens' genomes break the subtle regulation of microbial ecosystems (*microbiome*s) (Suchkov

The aforementioned studies have led to a reappraisal of Koch's postulates to announce that he had shown theoretically more then a hundred years before: one microbe causes one dis‐ ease. Interactions of microbial pathogens with specific tools of the patient's immune machi‐ nery in due the course of the infectious process give rise to different types of *micro-biocenosis* to manifest a broad range of activities of the microbiome-related pathogens including their

The key role of immune-related and microbial factor in CDIO pathogenesis is evidently proved today. However, a comparison of types of the patient's individual immune respons‐ es and the features of *microbial landscapes* with the clinical manifestations of the disease (*clini‐ cal pattern*) by singling out postinfectious clinical-and-immunological syndrome (PICIS) as a pathogenically oriented category and a fundamental term is appearing to be a more compli‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Cherepahina et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Mariya Bocharova, Murat Agirov,

ability to suppress the immune response.

cated objective to be cleared.

Sergey Suchkov

**1. Introduction**

et al., 2007).

http://dx.doi.org/10.5772/54128

**Chapter 7**

### **Microbiome and Autoimmunity**

Natalie Cherepahina, Zaur Shogenov, Mariya Bocharova, Murat Agirov, Jamilyia Tabaksoeva, Mikhail Paltsev and Sergey Suchkov

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54128

#### **1. Introduction**

Most of the currently known infections represent a persistent and stepwise development of the canonical chronic diseases of infectious origin (CDIO) associated with conditionally pathogenic microorganisms, i.e., viruses, intracellular parasites and other representatives of the endogenous microbiome, particularly, those manifesting with atypical properties. Pathogens are able to interact between themselves and to cause a disease, and the patho‐ gens' genomes break the subtle regulation of microbial ecosystems (*microbiome*s) (Suchkov et al., 2007).

The aforementioned studies have led to a reappraisal of Koch's postulates to announce that he had shown theoretically more then a hundred years before: one microbe causes one dis‐ ease. Interactions of microbial pathogens with specific tools of the patient's immune machi‐ nery in due the course of the infectious process give rise to different types of *micro-biocenosis* to manifest a broad range of activities of the microbiome-related pathogens including their ability to suppress the immune response.

The key role of immune-related and microbial factor in CDIO pathogenesis is evidently proved today. However, a comparison of types of the patient's individual immune respons‐ es and the features of *microbial landscapes* with the clinical manifestations of the disease (*clini‐ cal pattern*) by singling out postinfectious clinical-and-immunological syndrome (PICIS) as a pathogenically oriented category and a fundamental term is appearing to be a more compli‐ cated objective to be cleared.

© 2013 Cherepahina et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

As many authors established, for CDIО-related specific form of PICIS has clinical and prog‐ nostic significance and informative and thus predictive value, because, for sure, PICIS deter‐ mines to a considerable degree the extant of progressing and chronization of the underlying disease whilst determining risks of complications.

Unfortunately, the role of these mechanisms in the development and chronization of infec‐ tious diseases is still obscure, which deprives a doctor of the possibility to improve the cur‐ rently available models of CDIO chronization with the ultimate goal to develop the most advanced treatment-and-rehabilitation in a package of the preventive treatment protocols, and their implementation into routine and daily clinical practice (Paltsev et al., 2009a).

Microbiome and Autoimmunity http://dx.doi.org/10.5772/54128 135

The key role in the chronization and thus complications of such diseases, and thus the de‐

The microbial factor may promote development of a vast array of the associated immune disorders and thus forms of immune-mediated pathologies (PICIS), particularly, owing the microorganisms to gain, in the running course of the disease evolution, a large arsenal of in‐ trinsic tools enabling microbes to escape from attacks of immune-mediated weapon, to at‐ tenuate and the latter whilst getting them weakened or to influence the dynamics of the clinical manifestations of PICIS itself. Actually, the severity and duration of CDIO depends on the interaction between the microbiome and the antimicrobial immunity machinery (Palt‐

**2. PICIS and its role in the state-of-the-art model of immunopathogenesis**

The occurrence and thus incidence of these syndromes in CDIO patients differs statistically depending on the form of a type of the primary infection and the stage of the destructive inflammatory process in the targeted organs or tissues. For example, the initial (including *subclinical*) stages of any clinical courses of CDIO are usually concomitant with a predomi‐ nant (>50%) formation of PIFSIS, while the quotes of PIFAS and PIFASID do not exceed

This situation changes with further development and chronization of the infectious process and its transformation from a subclinical into the clinical stage, viz., the incidence of autoim‐ mune syndromes increases substantially. In intermediate stages of chronization, the quotes of PIFAS reach 50%, whilst in the final stages of chronization similar indices of PIFASID

It may thus be concluded that PIFSIS is not only the outcome of the infectious process, but, rather, the major factor responsible for provoking complications and raising thus a chronic

**1.** postinfectious secondary immunodeficiency syndrome (PIFSIS);

**3.** PIFAS coupled with PIFSIS (PIFASID) (Suchkov et al., 2004).

**2.** postinfectious autoimmune syndrome (PIFAS);

velopment of the associated immune disorders is played by:

**i.** properties of the infectious agent; **ii.** type of a patient's immune response.

**of chronic infectious diseases**

The PICIS variants include:

sev et al., 2009b).

20%.

reach 60%.

Postinfectious secondary immunodeficiency syndrome (PIFSI) is a *monosyndromal* and pre‐ dominant form of PICIS in patients with CDIO.

More than in a third of cases another form of PICIS could be formed and thus observed, ac‐ companying by postinfectious autoimmune syndrome (PIFA) reflecting the involvement of immune-mediated autoaggression, or PIFASID as an associated form of immune imbalance.

The susceptibility to one or another form of CDIO-associated PICIS depends on a number of genetically determined factors, which play a crucial role in formation of the patient's immune resources *via* cooperation between mechanisms of innate and adaptive immunity (Fig. 1).

**Figure 1.** Main features of innate and adaptive immunity

Unfortunately, the role of these mechanisms in the development and chronization of infec‐ tious diseases is still obscure, which deprives a doctor of the possibility to improve the cur‐ rently available models of CDIO chronization with the ultimate goal to develop the most advanced treatment-and-rehabilitation in a package of the preventive treatment protocols, and their implementation into routine and daily clinical practice (Paltsev et al., 2009a).

The key role in the chronization and thus complications of such diseases, and thus the de‐ velopment of the associated immune disorders is played by:

**i.** properties of the infectious agent;

As many authors established, for CDIО-related specific form of PICIS has clinical and prog‐ nostic significance and informative and thus predictive value, because, for sure, PICIS deter‐ mines to a considerable degree the extant of progressing and chronization of the underlying

Postinfectious secondary immunodeficiency syndrome (PIFSI) is a *monosyndromal* and pre‐

More than in a third of cases another form of PICIS could be formed and thus observed, ac‐ companying by postinfectious autoimmune syndrome (PIFA) reflecting the involvement of immune-mediated autoaggression, or PIFASID as an associated form of immune imbalance.

The susceptibility to one or another form of CDIO-associated PICIS depends on a number of genetically determined factors, which play a crucial role in formation of the patient's immune resources *via* cooperation between mechanisms of innate and adaptive immunity (Fig. 1).

disease whilst determining risks of complications.

134 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

dominant form of PICIS in patients with CDIO.

Note: NK, natural killer

**Figure 1.** Main features of innate and adaptive immunity

**ii.** type of a patient's immune response.

The microbial factor may promote development of a vast array of the associated immune disorders and thus forms of immune-mediated pathologies (PICIS), particularly, owing the microorganisms to gain, in the running course of the disease evolution, a large arsenal of in‐ trinsic tools enabling microbes to escape from attacks of immune-mediated weapon, to at‐ tenuate and the latter whilst getting them weakened or to influence the dynamics of the clinical manifestations of PICIS itself. Actually, the severity and duration of CDIO depends on the interaction between the microbiome and the antimicrobial immunity machinery (Palt‐ sev et al., 2009b).

### **2. PICIS and its role in the state-of-the-art model of immunopathogenesis of chronic infectious diseases**

The PICIS variants include:

The occurrence and thus incidence of these syndromes in CDIO patients differs statistically depending on the form of a type of the primary infection and the stage of the destructive inflammatory process in the targeted organs or tissues. For example, the initial (including *subclinical*) stages of any clinical courses of CDIO are usually concomitant with a predomi‐ nant (>50%) formation of PIFSIS, while the quotes of PIFAS and PIFASID do not exceed 20%.

This situation changes with further development and chronization of the infectious process and its transformation from a subclinical into the clinical stage, viz., the incidence of autoim‐ mune syndromes increases substantially. In intermediate stages of chronization, the quotes of PIFAS reach 50%, whilst in the final stages of chronization similar indices of PIFASID reach 60%.

It may thus be concluded that PIFSIS is not only the outcome of the infectious process, but, rather, the major factor responsible for provoking complications and raising thus a chronic relapsing course. The role of *progression* and *chronization* of the disease is determined, in a large measure, by the form of PICIS or, rather, of PIFAS and PIFASID, which reflect the manifestations and thus a pattern of postinfectious autoaggression (Suchkov et al., 2004).

over the microbiome itself, to switch off mechanisms of the control over the microbiome, or to initiate changes underlying the *resistance* phenomenon (Fig. 2). As a result of the antago‐ nistic interactions between a microbe and the antimicrobial immunity machinery, the im‐ mune response is either not elicited at all or is only partly activated giving rise to manifestations of PIFSIS, or progressing in a form provoking either PIFAS or PIFASID. This is concomitant with the formation of membrane films by clinical isolates of pathogenic mi‐

Microbiome and Autoimmunity http://dx.doi.org/10.5772/54128 137

In 30% of cases, the causative agent is not cultivated, but, rather, is converted into the *L*form, which does not exclude the presence of an "invisible" pathogen in the course of the instrumental and laboratory diagnostics procedures, and implies the latent progress of the infectious process. The latter presents particular interest because the conversion of some mi‐ croorganisms into the latent (*cryptic* or *sleeping*) form is one of the ways to chronization by

Conventional (predominantly, *gram*-negative) bacterial flora is detected in the majority of CDIO patients, though the results of the recent studies testify not only to the growth of *gram*-negative microorganisms, but also to a dramatic increase in the number of superinfec‐

The results of our recent studies suggest that the most frequently occurring human infec‐

**1.** susceptibility to relapsing infections provoked by pyogenic bacteria (*S. pneumoniae, S. aureus*, etc.) with a minimum *mimicking* resource increases dramatically in patients whose disorders are linked with antibody (Ab), complement and phagocytosis deficien‐

**2.** a particular susceptibility to viral and intracellular pathogens endowing with a high *mimicking* potential increases in patients with disturbances in primary cell-mediated im‐

Fluorescence has been used to show that the process of intraintestinal hatching of *Trichuris muris* is critically dependent on the attachment of an enterobacterium to the polar opercu‐ lum of the egg (*Panel A*). Hatched worms attach to the bowel wall and induce activation of type 2 helper T (Th2) cells, which in turn inhibit the proliferation and differentiation of type 17 helper T (Th17) and regulatory T (Treg) cells. In contrast, worm development was halted and specific immune responses were altered in mice that were depleted of gram-negative enterobacteria by antibiotics (*Panel B*), underscoring the influence of the intraintestinal envi‐

At the same time intracellular pathogens interfere in cell nucleus during DNA transcrip‐ tion. One of the key mechanisms by which microbes achieve the immunosuppressive ef‐

croorganisms escaping from the control of antimicrobial agents.

**4. Features of microbial spectra in CDIO patients**

tions may conventionally be divided into two main categories:

cy, PIFSI being the predominant form of PICIS;

escaping from the proper immune response.

tious pathogens of viral and parasitic origin.

ronment on intestinal immune responses.

munity (Fig. 3)

#### **3.** *Microbiome* **and its role in the state-of-the-art model of CDIO pathogenesis**

A crucial role in CDIO pathogenesis is played by primary and superinfectious factors en‐ dowed with the ability to generate large microbial associates. Therefore, the features of the patient's microbiome are key etiopathogenic links for realization of the infection-associated chronization phenomenon.

Notes: *Ag*, antigen

**Figure 2.** Scenarios of interactions between a microbial pathogen and the patient's antimicrobial immune response illustrating an escape from the immune supervision to survive

Moreover, in a course of their evolution microorganisms acquire a vast array of molecular and cellular tools enabling them to escape from the intrinsic control of the immune system over the microbiome itself, to switch off mechanisms of the control over the microbiome, or to initiate changes underlying the *resistance* phenomenon (Fig. 2). As a result of the antago‐ nistic interactions between a microbe and the antimicrobial immunity machinery, the im‐ mune response is either not elicited at all or is only partly activated giving rise to manifestations of PIFSIS, or progressing in a form provoking either PIFAS or PIFASID. This is concomitant with the formation of membrane films by clinical isolates of pathogenic mi‐ croorganisms escaping from the control of antimicrobial agents.

In 30% of cases, the causative agent is not cultivated, but, rather, is converted into the *L*form, which does not exclude the presence of an "invisible" pathogen in the course of the instrumental and laboratory diagnostics procedures, and implies the latent progress of the infectious process. The latter presents particular interest because the conversion of some mi‐ croorganisms into the latent (*cryptic* or *sleeping*) form is one of the ways to chronization by escaping from the proper immune response.

#### **4. Features of microbial spectra in CDIO patients**

relapsing course. The role of *progression* and *chronization* of the disease is determined, in a large measure, by the form of PICIS or, rather, of PIFAS and PIFASID, which reflect the manifestations and thus a pattern of postinfectious autoaggression (Suchkov et al., 2004).

A crucial role in CDIO pathogenesis is played by primary and superinfectious factors en‐ dowed with the ability to generate large microbial associates. Therefore, the features of the patient's microbiome are key etiopathogenic links for realization of the infection-associated

**Figure 2.** Scenarios of interactions between a microbial pathogen and the patient's antimicrobial immune response

Moreover, in a course of their evolution microorganisms acquire a vast array of molecular and cellular tools enabling them to escape from the intrinsic control of the immune system

**3.** *Microbiome* **and its role in the state-of-the-art model of CDIO**

136 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

**pathogenesis**

Notes: *Ag*, antigen

illustrating an escape from the immune supervision to survive

chronization phenomenon.

Conventional (predominantly, *gram*-negative) bacterial flora is detected in the majority of CDIO patients, though the results of the recent studies testify not only to the growth of *gram*-negative microorganisms, but also to a dramatic increase in the number of superinfec‐ tious pathogens of viral and parasitic origin.

The results of our recent studies suggest that the most frequently occurring human infec‐ tions may conventionally be divided into two main categories:

Fluorescence has been used to show that the process of intraintestinal hatching of *Trichuris muris* is critically dependent on the attachment of an enterobacterium to the polar opercu‐ lum of the egg (*Panel A*). Hatched worms attach to the bowel wall and induce activation of type 2 helper T (Th2) cells, which in turn inhibit the proliferation and differentiation of type 17 helper T (Th17) and regulatory T (Treg) cells. In contrast, worm development was halted and specific immune responses were altered in mice that were depleted of gram-negative enterobacteria by antibiotics (*Panel B*), underscoring the influence of the intraintestinal envi‐ ronment on intestinal immune responses.