Toll-like receptors TLR7 and TLR9 are both implicated in the activation of autoreactive B cells and other cell types associated with systemic lupus erythematosus (SLE) pathogenesis. However, Tlr9-/- autoimmune-prone strains paradoxically develop more severe disease. We have now leveraged the negative regulatory role of TLR9 to develop an inducible rapid-onset murine model of systemic autoimmunity that depends on T cell detection of a membrane-bound OVA fusion protein expressed by MHC class II+ cells, expression of TLR7, expression of the type I IFN receptor, and loss of expression of TLR9. These mice are distinguished by a high frequency of OVA-specific Tbet+, IFN-γ+, and FasL-expressing Th1 cells as well as autoantibody-producing B cells. Unexpectedly, contrary to what occurs in most models of SLE, they also developed skin lesions that are very similar to those of human cutaneous lupus erythematosus (CLE) as far as clinical appearance, histological changes, and gene expression. FasL was a key effector mechanism in the skin, as the transfer of FasL-deficient DO11gld T cells completely failed to elicit overt skin lesions. FasL was also upregulated in human CLE biopsies. Overall, our model provides a relevant system for exploring the pathophysiology of CLE as well as the negative regulatory role of TLR9.

Antibodies to multiple ovarian antigens have been proposed as markers of ovarian autoimmunity. The role of ovarian autoantibodies has been widely discussed in the pathophysiology of premature ovarian failure and unexplained infertility, but the autoantigens are yet to be identified. Three immunodominant ovarian autoantigens, α-actinin 4 (αACTN4), heat shock 70 protein 5 (HSPA5) and β-actin (ACTB), have been identified using anti-ovarian antibody-positive sera from women with idiopathic premature ovarian failure (n=50) and women undergoing IVF (n=695), using mass spectrometry. These autoantigens were subsequently validated using Western blot, immunohistochemistry and enzyme-linked immunosorbent assay. These autoantigens are localized to different components of the ovary such as the ooplasm of the oocyte, theca, granulosa, corpus luteum and zona pellucida. All the above antigens were found to be expressed in the ooplasm throughout follicular development. All the autoantigens are expressed specifically in the oocyte except αACTN4. The three autoantigens could contribute to the array of biomarkers to be used for developing specific and sensitive tests for diagnosis of women at risk of premature ovarian failure and IVF failure due to ovarian autoimmunity and could give an insight into the molecular mechanisms involved in the pathophysiology of these conditions.

Objective To establish importance of anti-ovarian antibodies (AOA) testing in infertile women. Design A clinical reproductive outcome comparative study between two groups of women undergoing IVF-ET. Group 1 consists of women tested positive for AOA, put on corticosteroid therapy, reverted to AOA negative and then taken up for IVF-ET. Group 2 were seronegative for AOA. Setting Major urban infertility reference centre and National research institute. Results AOA positive serum samples were sent periodically to re-investigate presence of AOA after corticosteroid therapy and women turned AOA negative were taken up for IVF-ET. Of the 70/138 women in group 1 who were treated with corticosteroids and turned seronegative for AOA, 22/70 were poor responders and needed donor oocyte-recipient cycles. Results demonstrated that fertilization and clinical pregnancy rates between both groups are comparable. Nevertheless, it is also observed that there is poor response to stimulation protocol, smaller number of oocytes retrieved and more spontaneous abortions in group 1 women. Hence not all outcomes following the treatment are comparable between the two groups. Usefulness of the test was established in two case studies. Conclusions AOA testing could be included in the battery of tests investigating and treating infertility.

Immunoproteomics using sera of women with ovarian autoimmune diseases such as primary ovarian insufficiency and IVF embryo transfer recruits led to identification of three proteins namely alpha actinin 4 (a-ACTN4), heat-shock 70 protein 5 (HSPA5), and actin beta (ACTB). This study deals with the establishment of a peptide ELISA for screening sera of antiovarian antibody (AOA)-positive patients and further delves into understanding the role of these three proteins in ovarian autoimmunity in a mouse model. Using in silico approach, antigenic peptides of these proteins were identified and used for peptide ELISA. ELISA results indicated that AOA-positive sera showed reactivity with only specific peptides. The functional significance of the dominant peptides was studied by active immunization of female mice with these peptides. All immunized mice generated high antibody titers and profound effect on ovaries with few primordial (2.4G0.1, 2.4G0.2, and 2G0.1), primary (2.4G0.5, 1.7G0.3, and 2.4G0.3), preantral (2.3G0.5, 3.4G0.3, and 2.9G0.3), antral (0.9G0.2, 1.6G0.8, and 2.3G0.6) follicles, and corpora lutea (2.8G0.8, 2.9G1.7, and 4.6G2.3), and increased number of atretic follicles (5.5G0.4, 4.9G1.8, and 7.5G1.0) in ACTN4-, HSPA5-, and ACTB-immunized mice compared with control animals (3.0G0.2, 3.5G0.6, 3G0.1, 3.6G0.2, 4.7G0.3, and 1.5G0.3) respectively. These mice when mated with fertile male mice showed an overall 25-43% reduction in fertility compared with controls. The data clearly suggest that the dominant antigenic epitopes of the three proteins play critical role in fertility and could possibly be the key autoimmune targets. These epitopes could be used to develop a more specific and sensitive diagnostic test for women with ovarian autoimmune diseases and to design therapy for disease management for reinstatement of ovarian function.

Cutaneous lupus erythematosus (CLE) is an autoimmune skin disease characterized by a strong IFN signature, normally associated with type I IFNs. However, increasing evidence points to an additional role for IFNγ, or at least a pathogenic T effector subset dependent on IFNγ, for disease progression. Nevertheless, Th2 effector subsets have also been implicated in CLE. We have now assessed the role of specific T cell subsets in the initiation and persistence of skin disease using a T cell-inducible murine model of CLE, dependent on KJ1-26 T cell recognition of an ovalbumin fusion protein. We found that only Th2-skewed cells, and not Th1-skewed cells, induced the development of skin lesions. However, we provide strong evidence that the Th2 disease-initiating cells convert to a more Th1-like functional phenotype in vivo by the time the skin lesions are apparent. This phenotype is maintained and potentiates over time, as T cells isolated from the skin, following a second induction of self-antigen, expressed more IFN-γ than T cells isolated at the time of the initial response. Transcriptional analysis identified additional changes in the KJ1-26 T cells at four weeks post injection, with higher expression levels of interferon stimulated genes (ISGs) including CXCL9, IRF5, IFIH1, and MX1. Further, injection of IFN-γ-/- T cells faied to induce skin disease in mice. We concluded that Th2 cells trigger skin lesion formation in CLE, and these cells switch to a Th1-like phenotype in the context of a TLR7-driven immune environment that is stable within the T cell memory compartment.

We postulate that the shared immunodominant peptide could be included in a peptide array to detect both HSAP5 and HSP90β autoantibodies for early diagnosis or prognosis of aPOI and customized glucocorticoid therapy for such subjects.

BCR signal strength plays a key role in the fate decision of B cells. Strong BCR signals promote plasma cell differentiation and weaker BCR signals drive germinal center B cell fate. Signals through toll-like receptors (TLRs) also influence B cell differentiation, but it is unclear how the BCR and TLR signaling cascades interface to modulate B cell responses and differentiation. To address this question, we used a BCR sdTg mouse model, AM14, in which all mature B cells have a naïve follicular phenotype. The AM14 BCR recognizes IgG2a-bound immune complexes (ICs) and AM14 B cell activation depends on TLR7 and/or TLR9. ICs formed by the anti-nucleosome antibody PL2-3 can activate AM14 B cells through either TLR7 or TLR9, allowing us to dissect signaling pathways unique to each TLR. We find that activation of AM14 TLR7-deficient B cells leads to sustained activation of the NF-κB pathway and p38 phosphorylation, subsequently leading to cell death. In contrast, TLR9-deficient AM14 B cells activated with ICs are unable to sustain the initial NF-κB and p38 signaling response. Intriguingly, these TLR9-deficient AM14 B cells differentiate into short-lived plasmablasts as characterized by increased levels of plasma cell associated proteins such as IRF-4, Blimp-1 and CD138. Plasma cell development is not observed in IC activated TLR7-deficient AM14 B cells. Our results suggest divergent roles for TLR7 and TLR9 in B cell activation and plasma cell differentiation.

Autoimmune diseases have gender bias with predominance in females, autoimmune infertility (AI) being no exception. This chapter will focus on AI in females with brief reference to the same in males. Autoimmune diseases have established protocols for detection and management of ensuing infertility, however similar protocols for unexplained infertility [tubal blockage, endometriosis, premature ovarian insufficiency (POI), undiagnosed underlying autoimmune disease (Sjögren's syndrome, IBS, celiac disease) and tubal blockage] are not established. Endometriosis and POI, in particular, have autoimmune etiology yet lack specific and sensitive biomarkers for accurate diagnosis. If autoantibodies are indeed diagnosed, then treatment regimen focuses on AI which has known adverse effects. The detection of natural antibodies as autoantibodies presents a viable alternative to organ specific biomarker panel for better management of AI.