A comparative study of antimicrobial activity of differently-synthesized chitosan nanoparticles against bovine mastitis pathogens.

The greatest concern in dairy farming nowadays is bovine mastitis (BM), which results mainly from bacterial colonization of the mammary gland. Antibiotics are the most widely used strategy for its prevention and treatment, but overuse has led to growing antimicrobial resistance. Pathogens have also developed other mechanisms to persist in the udder, such as biofilm formation and internalization into bovine epithelial cells. New therapies are therefore needed to reduce or replace antibiotic therapies. In a previous study, we found that chitosan nanoparticles (Ch-NPs) have considerable potential for the treatment of BM. The aim of the present study was to evaluate the antimicrobial activity of differently-synthesized Ch-NPs against BM pathogens and their toxicity in bovine cells in vitro, to further explore the attributes of Ch-NPs for the prevention and treatment of intramammary infections. We also looked into their ability to inhibit biofilm formation and prevent the internalization of S. aureus into mammary epithelial cells. Finally, since an interesting approach for BM prevention is to enhance the host's immune response, we studied whether Ch-NPs could promote the release of pro-inflammatory cytokines in mammary epithelial cells. The results reveal that the bactericidal effect of Ch-NPs on BM pathogens and their ability to inhibit biofilm formation are size-dependent, with smaller particles being more efficient. In contrast, their effect on the viability of the cell lines is not size-dependent and all samples tested were non-toxic. The smallest Ch-NPs successfully prevented the internalization of S. aureus into the cells, but did not promote the production of pro-inflammatory cytokines. These findings make it possible to conclude that Ch-NPs are a great bactericidal agent which can prevent the main mechanisms developed by BM pathogens to persist in the udder.

Patients with chronic prostatitis/chronic pelvic pain syndrome show T helper type 1 (Th1) and Th17 self-reactive immune responses specific to prostate and seminal antigens and diminished semen quality.

OBJECTIVES: To assess the presence of self-reactive immune responses to seminal and prostate antigens (PAg), biomarkers of inflammation of the male genital tract, and semen quality parameters in patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). PATIENTS, SUBJECTS AND METHODS: Peripheral blood and semen samples were collected from patients with CP/CPPS and age-matched healthy control volunteers. We analysed the lymphoproliferative responses of peripheral blood mononuclear cells (PBMC) to different seminal plasma (SP)-derived and purified PAg, serum autoantibodies specific to PAg, leucocyte subpopulations, and inflammatory cytokines in semen, sperm apoptosis/necrosis, and semen quality parameters. RESULTS: Significantly greater PBMC proliferative responses specific to PAg, with elevated secretion of interferon (IFN)γ and interleukin (IL)-17, were detected in the patients with CP/CPPS vs the controls. Moreover, the patients with CP/CPPS had significantly greater serum immunoglobulin G immune reactivity to SP proteins, such as prostate-specific antigen and prostatic acid phosphatase, than the controls. Inflammation of the male genital tract was exemplified by high levels of IFNγ, IL-17, IL-1ß and IL-8, as well as higher counts of leukocytes, mainly CD4 T lymphocytes and macrophages, in the semen. In addition, this local inflammation was associated with an overall diminished semen quality, i.e., reduced sperm concentration, motility and viability; and higher levels of sperm apoptosis/necrosis in patients with CP/CPPS vs controls. CONCLUSION: Patients with CP/CPPS show T helper type 1 (Th1) and Th17 immune responses specific to PAg associated with chronic inflammation of the male genital tract and reduced semen quality. These immune responses may underlie the induction and development of chronic pelvic pain and inflammation of the male genital tract, which in turn could alter normal prostate functioning and impair semen quality.

Implications of prostate inflammation on male fertility.

The prostate is the seat of three major causes of morbidity: benign prostatic hyperplasia, prostate cancer and prostatitis, three conditions in which inflammation has been implicated. A state of inflammation of the prostate gland, originally incited by an infection, an autoimmune response, a neurogenic stimulus or another trigger may have consequences on prostate functionality. In fact, male fertility depends intrinsically on the content of prostatic fluid factors secreted by the prostatic epithelium. Taking into account that the prostate gland is the major male accessory gland that exerts essential functions for male fertility, a state of local inflammation can alter male fertility by either directly impairing sperm quality or, indirectly, by causing prostate dysfunction. In the present review, we summarise the current knowledge regarding prostatitis due to well-known infections such as Escherichia coli, Chlamydia trachomatis and other commonly identified microorganisms focusing on inflammatory markers detected during these infections and seminal quality and male fertility alterations reported. We also focused on type III prostatitis or chronic nonbacterial prostatitis/chronic pelvic pain syndrome, of unknown aetiology, in which inflammation of an autoimmune origin, neurogenic stimuli or another trigger have been proposed and fertility alterations reported.

Chronic Pelvic Pain Development and Prostate Inflammation in Strains of Mice With Different Susceptibility to Experimental Autoimmune Prostatitis.

BACKGROUND: Experimental autoimmune prostatitis (EAP) is an autoimmune inflammatory disease of the prostate characterized by peripheral prostate-specific autoimmune responses associated with prostate inflammation. EAP is induced in rodents upon immunization with prostate antigens (PAg) plus adjuvants and shares important clinical and immunological features with the human disease chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). METHODS: EAP was induced in young NOD, C57BL/6, and BALB/c male mice by immunization with PAg plus complete Freund́s adjuvant. Tactile allodynia was assessed using Von Frey fibers as a measure of pelvic pain at baseline and at different time points after immunization. Using conventional histology, immunohistochemistry, FACS analysis, and protein arrays, an interstrain comparative study of prostate cell infiltration and inflammation was performed. RESULTS: Chronic pelvic pain development was similar between immunized NOD and C57BL/6 mice, although the severity of leukocyte infiltration was greater in the first case. Coversely, minimal prostate cell infiltration was observed in immunized BALB/c mice, who showed no pelvic pain development. Increased numbers of mast cells, mostly degranulated, were detected in prostate samples from NOD and C57BL/6 mice, while lower total counts and resting were observed in BALB/c mice. Prostate tissue from NOD mice revealed markedly increased expression levels of inflammatory cytokines, chemokines, adhesion molecules, vascular endothelial growth factor, and metalloproteinases. Similar results, but to a lesser extent, were observed when analyzing prostate tissue from C57BL/6 mice. On the contrary, the expression of the above mediators was very low in prostate tissue from immunized BALB/c mice, showing significantly slight increments only for CXCL1 and IL4. CONCLUSIONS: Our results provide new evidence indicating that NOD, C57BL/6, and BALB/c mice develop different degrees of chronic pelvic pain, type, and amount of prostate cell infiltration and secretion of inflammatory mediators. Our results corroborate and support the notion that mice with different genetic background have different susceptibility to EAP induction. Prostate 77:94-104, 2017. © 2016 Wiley Periodicals, Inc.

Chronic Infection of the Prostate by Chlamydia muridarum Is Accompanied by Local Inflammation and Pelvic Pain Development.

BACKGROUND: Chlamydia trachomatis urogenital infections are the leading cause of sexually transmitted bacterial infections. Although the prevalence of chlamydial infection is similar in men and women, current research is mainly focused on women, neglecting the study of male genital tract infections. We, therefore, investigated Chlamydia infection in the rodent male genital tract. MATERIALS AND METHODS: Male NOD and C57BL/6 mice were inoculated in the meatus urethra with C. muridarum. Bacterial DNA, leukocyte infiltration of male genital tract tissues, pelvic pain, and Chlamydia-specific immune responses were analyzed at different time points. RESULTS AND CONCLUSIONS: The inoculation of C. muridarum in the meatus urethra of male mice resulted in an ascending and widely disseminated infection of the male genital tract. C. muridarum remained longer and with the highest bacterial burdens in the prostate, thus showing a special tropism for this organ. Infection caused leukocyte infiltration, mainly composed by neutrophils, and also induced early pelvic pain development that rapidly dropped and resolved as the infection became chronic. Bacterial load and leukocyte infiltration was observed in all prostate lobes, although dorsolateral prostate was the most affected lobe. Interestingly, immune responses induced by both mice strains were characterized by the production of high levels of IL-10 during early stages of the infection, with highest and sustained levels observed in NOD mice, which showed to be less efficient in clearing the infection. Chronic infection of the prostate accompanied by local inflammation and pelvic pain development described herein have important implications for the improvement of the diagnosis and for the design of new efficient therapies. Prostate 77:517-529, 2017. © 2017 Wiley Periodicals, Inc.

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.

Differences in T regulatory cells between mouse strains frequently used in immunological research: Treg cell quantities and subpopulations in NOD, B6 and BALB/c mice.

Foxp3+ Regulatory T cells (Tregs) are essential for the maintenance of tolerance to self. Therefore, it is expected that lower numbers and/or less than optimal function could impact on the functioning of the immune system, and thereby contributing to the development of autoimmune diseases. In the present report, by comparing Tregs from most frequently used mouse strains in immunological research (C57BL/6 (B6), BALB/c and NOD), we provide evidence showing that the NOD mouse strain, highly predisposed to develop autoimmune responses, exhibit a generalized decreased in Tregs counts with enhanced proportions of CD44hiCD62Llow Tregs when compared with BALB/c mice. No major differences were observed in Helios+ or Helios- Tregs between strains. The expression of CXCR3, CCR5 and CCR6 on Tregs from all strains showed minor proportions of CXCR3+ and CCR5+ cells in NOD Tregs. Naïve CD4+CD25- T cells from NOD mice also showed decreased capacity to induce in vitro iTregs when compared with B6 and BALB/c mice. Lower expression of molecules involved in Treg suppressor mechanisms such as CD25, LAP-1, CD39 and PD-1 was observed both in NOD iTregs and Tregs from lymph nodes of NOD mice. Moreover, in vitro assays showed that Tregs from NOD mice exhibited reduced ability to suppress proliferation of CD4+CD25- responder T cells when compared with B6 and BALB/c mice. Major differences were consistently observed between NOD and BALB/c mice, whereas no major differences were found for many of the analyzed parameters between the NOD and B6 mice, suggesting that highly and mildly autoimmune prone mouse strains may share some Tregs features. On the contrary, BALB/c Tregs were in major quantities, expressed higher levels of Foxp3 and exhibited more potent ability to inhibit effector T cell proliferation, data that could be related to its natural resistance to the induction of different experimental autoimmune conditions. Altogether our results demonstrate a generalized Treg cell dysfunction in NOD mice, a strain characterized by its high predisposition to develop spontaneous and induced autoimmune diseases.

IL-10 Producing B Cells Dampen Protective T Cell Response and Allow Chlamydia muridarum Infection of the Male Genital Tract.

A significant proportion of individuals develop chronic, persistent and recurrent genital tract infections with Chlamydia trachomatis, which has been attributed to the numerous strategies that the bacterium uses to subvert host immune responses. Animal chlamydia models have demonstrated that protective immune response is mediated by CD4+ Th1 cytokine responses. Herein, we demonstrate that early after infecting the male genital tract, C. muridarum triggers the production of IL-10 by splenic and lymph node cells. In addition, C. muridarum triggers IL-6 and TNFα secretion. Data obtained from in vitro and in vivo experiments revealed B cells as the major IL-10 contributors. Indeed, purified B cells produced high amounts of IL-10 and also exhibited enhanced expression of inhibitory molecules such as CD39, PD-L1 and PD1 after C. muridarum stimulation. In vitro experiments performed with sorted cell subsets revealed that Marginal Zone B cells were the main IL-10 producers. In vitro and in vivo studies using TLR-deficient mice indicated that TLR4 signaling pathway was essential for IL-10 production. In addition, in vivo treatments to neutralize IL-10 or deplete B cells indicated that IL-10 and B cells played a significant role in delaying bacterial clearance ability. Moreover, the latter was confirmed by adoptive cell transfer experiments in which the absence of IL-10-producing B cells conferred the host a greater capability to induce Th1 responses and clear the infection. Interestingly, NOD mice, which were the least efficient in clearing the infection, presented much more Marginal Zone B counts and also enhanced TLR4 expression on Marginal Zone B cells when compared to B6 and BALB/c mice. Besides, treatment with antibodies that selectively deplete Marginal Zone B cells rendered mice more capable of inducing enhanced IFNγ responses and clearing the infection. Our findings suggest that B cells play a detrimental role in C. muridarum infection and that activation by innate receptors like TLR4 and IL-10 production by these cells could be used by Chlamydia spp. as a strategy to modulate the immune response establishing chronic infections in susceptible hosts.

Chitosan and cloxacillin combination improve antibiotic efficacy against different lifestyle of coagulase-negative Staphylococcus isolates from chronic bovine mastitis.

Bovine mastitis affects the health of dairy cows and the profitability of herds worldwide. Coagulase-negative staphylococci (CNS) are the most frequently isolated pathogens in bovine intramammary infection. Based on the wide range of antimicrobial, mucoadhesive and immunostimulant properties demonstrated by chitosan, we have evaluated therapy efficiency of chitosan incorporation to cloxacillin antibiotic as well as its effect against different bacterial lifestyles of seven CNS isolates from chronic intramammary infections. The therapeutic effects of combinations were evaluated on planktonic cultures, bacterial biofilms and intracellular growth in mammary epithelial cells. We found that biofilms and intracellular growth forms offered a strong protection against antibiotic therapy. On the other hand, we found that chitosan addition to cloxacillin efficiently reduced the antibiotic concentration necessary for bacterial killing in different lifestyle. Remarkably, the combined treatment was not only able to inhibit bacterial biofilm establishment and increase preformed biofilm eradication, but it also reduced intracellular bacterial viability while it increased IL-6 secretion by infected epithelial cells. These findings provide a new approach to prophylactic drying therapy that could help to improve conventional antimicrobial treatment against different forms of bacterial growth in an efficient, safer and greener manner reducing multiresistant bacteria generation and spread.

Immunological Mechanisms Underlying Chronic Pelvic Pain and Prostate Inflammation in Chronic Pelvic Pain Syndrome.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is the most common urologic morbidity in men younger than 50 years and is characterized by a diverse range of pain and inflammatory symptoms, both in type and severity, that involve the region of the pelvis, perineum, scrotum, rectum, testes, penis, and lower back. In most patients, pain is accompanied by inflammation in the absence of an invading infectious agent. Since CP/CPPS etiology is still not well established, available therapeutic options for patients are far from satisfactory for either physicians or patients. During the past two decades, chronic inflammation has been deeply explored as the cause of CP/CPPS. In this review article, we summarize the current knowledge regarding immunological mechanisms underlying chronic pelvic pain and prostate inflammation in CP/CPPS. Cumulative evidence obtained from both human disease and animal models indicate that several factors may trigger chronic inflammation in the form of autoimmunity against prostate, fostering chronic prostate recruitment of Th1 cells, and different other leukocytes, including mast cells, which might be the main actors in the consequent development of chronic pelvic pain. Thus, the local inflammatory milieu and the secretion of inflammatory mediators may induce neural sensitization leading to chronic pelvic pain development. Although scientific advances are encouraging, additional studies are urgently needed to establish the relationship between prostatitis development, mast cell recruitment to the prostate, and the precise mechanisms by which they would induce pelvic pain.

IL-17 is not essential for inflammation and chronic pelvic pain development in an experimental model of chronic prostatitis/chronic pelvic pain syndrome.

Pain and inflammation in the absence of infection are hallmarks in chronic prostatitis and chronic pelvic pain syndrome (CP/CPPS) patients. The etiology of CP/CPPS is unclear, and autoimmunity has been proposed as a cause. Experimental autoimmune prostatitis (EAP) models have long been used for studying CP/CPPS. Herein, we studied prostate inflammation induction and chronic pelvic pain development in EAP using IL-12p40-KO, IL-4-KO, IL-17-KO, and wild-type (C57BL/6) mice. Prostate antigen (PAg) immunization in C57BL/6 mice induced specific Th1 and Th17 immune responses and severe prostate inflammation and cell infiltration, mainly composed of CD4 T cells and macrophages. Moreover, chronic pelvic pain was evidenced by increased allodynia responses. In immunized IL-17-KO mice, the presence of a prominent PAg-specific Th1 immune response caused similar prostate inflammation and chronic pelvic pain. Furthermore, markedly high PAg-specific Th1 immune responses, exacerbated prostate inflammation, and chronic pelvic pain were detected in immunized IL-4-KO mice. Conversely, immunized IL-12p40-KO mice developed PAg-specific Th2 immune responses, characterized by high IL-4 secretion and neither infiltration nor damage in the prostate. As observed in wild-type control animals, IL12p40-KO mice did not evidence tactile allodynia responses. Our results suggest that, as in patients, chronic pelvic pain is a consequence of prostate inflammation. After PAg immunization, a Th1-associated immune response develops and induces prostate inflammation and chronic pelvic pain. The absence of Th1 or Th2 cytokines, respectively, diminishes or enhances EAP susceptibility. In addition, IL-17 showed not to be essential for pathology induction and chronic pelvic pain development.

Regulatory T cells control strain specific resistance to Experimental Autoimmune Prostatitis.

Susceptibility to autoimmune diseases results from the encounter of a complex and long evolved genetic context with a no less complex and changing environment. Major actors in maintaining health are regulatory T cells (Treg) that primarily dampen a large subset of autoreactive lymphocytes escaping thymic negative selection. Here, we directly asked whether Treg participate in defining susceptibility and resistance to Experimental Autoimmune Prostatitis (EAP). We analyzed three common laboratory strains of mice presenting with different susceptibility to autoimmune prostatitis upon immunization with prostate proteins. The NOD, the C57BL/6 and the BALB/c mice that can be classified along a disease score ranging from severe, mild and to undetectable, respectively. Upon mild and transient depletion of Treg at the induction phase of EAP, each model showed an increment along this score, most remarkably with the BALB/c mice switching from a resistant to a susceptible phenotype. We further show that disease associates with the upregulation of CXCR3 expression on effector T cells, a process requiring IFNγ. Together with recent advances on environmental factors affecting Treg, these findings provide a likely cellular and molecular explanation to the recent rise in autoimmune diseases incidence.