Ruben D. Motrich

Autoimmune Prostatitis: State of the Art

The prostate is one of the main male sex accessory glands and the target of many pathological conditions affecting men of all ages. Pathological conditions of the prostate gland range from infections, chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) of a still unknown aetiology to benign hyperplasia and cancer. CP/CPPS is one of the most prevalent diseases in the urologic clinic and affects men younger than 50 years old. A significant advance in the understanding of CP/CPPS was made when an autoimmune response against prostate antigens was revealed in a considerable number of patients. During the last 30 years, extensive work has been done regarding the development and characterization of different rodent models of experimental autoimmune prostatitis (EAP). It has been demonstrated that tolerance to prostate antigens can be disrupted in some strains of rats and mice and cellular and humoral responses to prostate antigens are elicited. A Th1 pattern has been described and the cellular response seems to be the major pathogenic mechanism involved. Immune cells infiltrate the gland and induce prostate lesions. The genetic background and hormonal imbalance are factors that could contribute to the onset of the disease in susceptible young males. Moreover, spontaneous autoimmune prostatitis could also occur with advanced age in susceptible strains. In this review, we summarize the current knowledge regarding rodent models of EAP and the immunological alterations present in CP/CPPS patients. We also discuss the reliability of these experimental approaches as genuine tools for the study of human disease.

Prostate epithelial cells can act as early sensors of infection by up-regulating TLR4 expression and proinflammatory mediators upon LPS stimulation

Despite the prevalence of prostate disease, little is known about the immunobiology of the prostate and its contribution to disease. The main goal of this work was to investigate how prostate epithelial cells deal with inflammatory stimuli. To this aim, we stimulated a rat prostate epithelial cell line [metastasis‐lung (MAT‐LU)] or rat primary epithelial cells with lipopolysaccharide (LPS). Prostate epithelial cells constitutively express significant levels of Toll‐like receptor 4 (TLR4) and CD14 mRNA. TLR2 transcription could also be demonstrated, suggesting that these cells could recognize a broader spectrum of microbial molecular patterns. TLR4, TLR2, and CD14 proteins were also detected, although not at the cell surface but intracellularly. Prostate epithelial cells not only express these receptors, but they are also able to respond to LPS, and LPS‐stimulated MAT‐LU cells activate nuclear factor‐κB transcription factor, induce the expression of inducible nitric oxide (NO) synthase, and secrete NO. Even more, numerous chemokine genes are up‐regulated or induced in this response. Our results clearly demonstrate that prostate epithelial cells are fully competent to respond. The fact that they express TLR4 and TLR2 intracellularly suggests the presence of regulatory mechanisms, which once overcome, could turn these cells into active players of the innate immunity, capable of initiating an inflammatory response.

Pathogenic Consequences in Semen Quality of an Autoimmune Response against the Prostate Gland: From Animal Models to Human Disease

We have recently proposed an autoimmune etiology in ∼35% of chronic nonbacterial prostatitis patients, the most frequent form of prostatitis observed, because they exhibit IFN-γ-secreting lymphocytes specific to prostate Ags. Interestingly, this particular group of patients, but not the rest of chronic nonbacterial prostatitis patients, also presented striking abnormalities in their semen quality. In this work, we use an experimental animal model of autoimmune prostatitis on Wistar rats developed in our laboratory to investigate when, where, and how sperm cells from autoimmune prostatitis individuals are being damaged. As in patients, a marked reduction in sperm concentration, almost null sperm motility and viability, and an increased percentage of apoptotic spermatozoa were detected in samples from animals with the disease. Prostate-specific autoantibodies as well as elevated levels of NO, TNF-α, and IFN-γ were also detected in their seminal plasma. In contrast, epididymal spermatozoa remain intact, indicating that sperm damage occurs at the moment of joining of prostate secretion to sperm cells during ejaculation. These results were further supported by experiments in which mixture of normal sperm cells with autoimmune seminal plasma were performed. We hypothesize that sperm damage in experimental autoimmune prostatitis can be the consequence of an inflammatory milieu, originally produced by an autoimmune response in the prostate; a diminished prostate functionality, evidenced by reduced levels of citric acid in semen or by both mechanisms simultaneously. Once more, we suggest that autoimmunity to prostate may have consequences on fertility.

Cow's milk stimulated lymphocyte proliferation and TNFα secretion in hypersensitivity to cow's milk protein

Although there is no reliable single laboratory test available for the diagnosis of cows milk allergy, if an allergic mechanism is suspected, a number of laboratory studies may be useful in delineating specific proteins responsible for these disorders. In the current study we analyzed in vitro lymphocyte proliferation assays, specific secretion of TNFalpha in supernatant cultures and specific IgE, IgG, and IgA in a group of patients with hypersensitivity to cows milk antigens. The stimulation index against a cows milk antigen mixture, alpha-lactalbumin, beta-lactoglobulin, and casein was significant higher in the group of patients maintained on cows milk-free diet for less than 4 months compared with the values observed in the control group and in the group of patients without a close contact to cows milk proteins. A significant increase in TNF-alpha secretion was observed in supernatants from patients with close contact to cows milk (CM). Specific IgE was detected in 59.3% of the patients, with higher specific IgE levels in patients who were not positive for the proliferation assay, suggesting a clear difference in the two mechanisms proposed as effectors in this disease. No differences in specific IgG and IgA levels were observed between the patient group and the control group, with a great dispersion among individuals in all groups tested. We conclude that a combination of the assays tested in this study, such as proliferative assay of peripheral blood mononuclear cells to CM, the quantitation of TNFalpha in culture supernatants, and serum specific IgE determination, are useful laboratory tests to identify cows milk allergy among patients with immediate and non immediate adverse reactions, reducing the need for food allergen challenges in young children.

Expression of CXCR3 on Specific T Cells Is Essential for Homing to the Prostate Gland in an Experimental Model of Chronic Prostatitis/Chronic Pelvic Pain Syndrome

Experimental autoimmune prostatitis (EAP) is considered a valid model for the human disease chronic prostatitis/chronic pelvic pain syndrome. In this report, we analyzed phenotypic characteristics of T cells that gain access to the prostate and induce leukocyte recruitment in mice with different susceptibility to EAP. After EAP induction, NOD mice developed a specific cellular response characterized by a mixed Th1/Th17 pattern with specific T cells mainly expressing CXCR3 that infiltrated and damaged the prostate. In contrast, BALB/c mice, as well as NOD-IFN-γ−/−, exhibited only Th17 cells mainly expressing CCR6 that were not capable of infiltrating the prostate gland. Adoptive transfer experiments of T cells from NOD or NOD–IFN-γ−/− mice to NOD-SCID recipients showed that only T cells from NOD mice successfully infiltrated the prostate. However, after “in vitro” or “in vivo” treatment with rIFN-γ, T cells from NOD–IFN-γ−/− mice became capable of homing to the prostate and induced leukocyte recruitment. Chemokine levels in prostate tissue from NOD mice showed increased expression levels of CXCR3 ligands. Additional experiments using adoptive transfer of sorted CXCR3+CD3+ T cells or administrating a CXCR3 antagonist treatment confirmed these previous results. Altogether, our results demonstrate that the expression of CXCR3 on effector T cells is essential for their homing to the prostate gland in EAP. CXCR3 emerges as a potential therapeutic target to control chronic prostatitis/chronic pelvic pain syndrome.

Transglutaminase 4 as a prostate autoantigen in male subfertility

TGM4 is a male-specific autoantigen for prostatitis associated with autoimmune polyendocrine syndrome type 1. AIREing out autoimmunity Patients with autoimmune polyendocrine syndrome type 1(APS1) experience dysfunction in multiple endocrine glands due to mutations in the AIRE gene, which helps promote immune tolerance. These patients frequently are infertile; female infertility can be explained by autoimmune ovarian failure, but the causes of male infertility have remained unclear. Now, Landegren et al. report that the prostatic secretory molecule tranglutaminase 4 (TGM4) is a male-specific autoantigen in APS1 patients that could contribute to subfertility. They found autoantibodies to TGM4 in APS1 patients beginning at puberty, and confirmed in AIRE-deficient mice that TGM4 autoantibodies lead to a destructive prostatitis. These data could help explain infertility in male APS1 patients. Autoimmune polyendocrine syndrome type 1 (APS1), a monogenic disorder caused by AIRE gene mutations, features multiple autoimmune disease components. Infertility is common in both males and females with APS1. Although female infertility can be explained by autoimmune ovarian failure, the mechanisms underlying male infertility have remained poorly understood. We performed a proteome-wide autoantibody screen in APS1 patient sera to assess the autoimmune response against the male reproductive organs. By screening human protein arrays with male and female patient sera and by selecting for gender-imbalanced autoantibody signals, we identified transglutaminase 4 (TGM4) as a male-specific autoantigen. Notably, TGM4 is a prostatic secretory molecule with critical role in male reproduction. TGM4 autoantibodies were detected in most of the adult male APS1 patients but were absent in all the young males. Consecutive serum samples further revealed that TGM4 autoantibodies first presented during pubertal age and subsequent to prostate maturation. We assessed the animal model for APS1, the Aire-deficient mouse, and found spontaneous development of TGM4 autoantibodies specifically in males. Aire-deficient mice failed to present TGM4 in the thymus, consistent with a defect in central tolerance for TGM4. In the mouse, we further link TGM4 immunity with a destructive prostatitis and compromised secretion of TGM4. Collectively, our findings in APS1 patients and Aire-deficient mice reveal prostate autoimmunity as a major manifestation of APS1 with potential role in male subfertility.

Impact of vitamin D receptor activity on experimental autoimmune prostatitis

Chronic non bacterial prostatitis is a chronic inflammatory syndrome. Its etiology and physiopathology are unclear and treatments are empirical and ineffective in most cases. Autoimmunity has been proposed as an etiology. In the present report, we investigated the impact of vitamin D receptor silencing, by use of VDR-KO NOD mice and the immune-modulating effect of the vitamin D3 analog TX527 on the development of Experimental Autoimmune Prostatitis in NOD mice. VDR-KO NOD mice developed a more aggressive form of autoimmune prostatitis characterized by a greater lymphoproliferative response against prostate antigen in vitro (6.92+/-4.77 vs. 2.47+/-0.41 21 days after disease induction, p<0.05) and higher levels of specific INFgamma secretion (471+/-6 vs. 386+/-5pg/ml, p<0.01). This was accompanied in vivo by more severe lesions and augmented mononuclear cell infiltration in the prostate gland. On the other hand, although analog-treated mice showed a significant reduction in the spleen T-cell specific proliferative response against prostate antigen in vitro, no effect on disease development was observed. We conclude that vitamin D receptor modulation holds the promise of interfering with autoimmune prostatitis. Introduction of more powerful analogs, or combinations with anti-T-cell reagents may represent therapeutic solutions for these group of patients.

Male Rodent Genital Tract Infection With Chlamydia Muridarum: Persistence in the Prostate Gland That Triggers Self-Immune Reactions in Genetically Susceptible Hosts

PURPOSE We investigated Chlamydia trachomatis infection and its pathogenic consequences in the male rodent genital tract. MATERIALS AND METHODS Male rats were inoculated in the meatal urethra with Chlamydia muridarum. We sought bacterial DNA at early and late times after inoculation in different parts of the male genital tract. Histological alterations and the immune response against prostate antigens were analyzed. RESULTS Male rats showed ascending infection with wide dissemination of bacteria in the genital tract at an early time point after inoculation. At later stages bacteria persisted only in some parts of the genital tract and in the prostate gland. C. muridarum was also detected in semen in a high proportion of rats irrespective of an acute or chronic stage of infection. Histological alterations that accompanied C. muridarum were especially observed in the prostate and mainly composed of CD3+ cell infiltration. Positive humoral and cellular responses against prostate antigens were noted in a considerable number of infected rats. NOD mice, an autoimmune, prostatitis prone strain, showed a similar pattern with C. muridarum in the prostate of 100% of infected mice, which was again accompanied by mononuclear cell infiltration and antibodies against prostate antigens at early and late times after inoculation. CONCLUSIONS Results reveal that C. muridarum infects the male rodent genitourinary tract with special persistence in the prostate gland, where it causes chronic inflammation that in turn may act as a trigger factor for self-immune reactions in susceptible hosts.

Effects of autoimmunity to the prostate on the fertility of the male rat

OBJECTIVE To study the putative direct consequences of autoimmunity to the prostate on male semen quality and fertility potential. DESIGN Experimental animal study. SETTING Immunology, CIBICI-CONICET, National University of Cordoba, Argentina. ANIMAL(S) Twelve male and 24 female Wistar rats. INTERVENTION(S) Autoimmune prostatitis was induced following a standard protocol. MAIN OUTCOME MEASURE(S) Citric acid, zinc, sperm vitality, reactive oxygen species levels, total antioxidant capacity, DNA fragmentation, and lipid peroxidation were analyzed in semen samples. Mating studies were also performed, and different fertility parameters (potency, fecundity index, fertility index, fetus size, and preimplantation and postimplantation embryo loss) were assayed. RESULT(S) Citric acid and zinc levels were decreased in seminal samples from autoimmune rats. Increased reactive oxygen species values and lower levels of total antioxidant capacity were shown in semen samples from autoimmune animals. Moreover, decreased levels of sperm vitality, high percentages of damaged DNA sperm cells, and elevated lipid peroxidation levels also were shown in semen from autoimmune rats. A significant decrease in the fertility index and augmented percentages of embryo loss were observed in the autoimmune group. CONCLUSION(S) An autoimmune response to the prostate impairs its functionality and induces important alterations in seminal oxidative balance, producing higher levels of lipid peroxidation and sperm DNA damage. Moreover, autoimmunity to the prostate gland seriously compromises male fertility.

Crucial Role of Interferon-γ in Experimental Autoimmune Prostatitis

PURPOSE An autoimmune etiology is proposed in some patients with chronic nonbacterial prostatitis since they show interferon-gamma secreting lymphocytes specific to prostate antigens in the periphery and increased interferon-gamma in seminal plasma. We investigated the involvement of interferon-gamma in an animal model of autoimmune prostatitis. MATERIALS AND METHODS Experimental autoimmune prostatitis was studied in the no-obese diabetic and C57Bl/6 (Harlan, Zeist, The Netherlands) susceptible mouse strains, and in the IRF-1 KO and STAT-1 KO mouse strains deficient in transcription factors involved in interferon-gamma signaling. RESULTS Experimental autoimmune prostatitis was characterized by prostate specific interferon-gamma secreting cells in the periphery and by T-helper 1 related cytokines in the target organ. Increased interferon-gamma and interleukin-12 were observed in the prostate of autoimmune animals while interleukin-10 and interleukin-4 were decreased and unaltered, respectively. The absence of transcription factors involved in the interferon-gamma signaling cascade, IRF-1 and STAT-1, made mice resistant to experimental autoimmune prostatitis. IRF-1 KO and STAT-1 KO mice immunized with prostate antigens did not show infiltration or alterations in the prostate. They did not have the typical prostate specific autoimmune response and showed decreased interferon-gamma, interleukin-12 and interleukin-10, and augmented interleukin-4 in the prostate. CONCLUSIONS Our results argue for a crucial role of interferon-gamma as a key factor in the pathogenesis of the disease. Intense research is promptly required to identify the pathogenic mechanisms underlying chronic prostatitis/chronic pelvic pain syndrome to find a more rational therapy.