The Protective Role of pVHL in Imiquimod-Induced Psoriasis-like Skin Inflammation.

Psoriasis is a chronic inflammatory disease distinguished by an excessive proliferation and abnormal differentiation of keratinocytes. Immune cells, such as T lymphocytes and neutrophils, and inflammatory cytokines, such as Tumor Necrosis Factor-α (TNF-α) and interleukin 17 (IL-17), are essential for maintaining psoriatic lesions. Additionally, a hypoxic milieu present in the skin promotes the expression of transcriptional factor hypoxia-inducible factor-1 alpha (HIF-1α). This protein regulates the expression of angiogenic and glycolytic factors, such as vascular endothelial grown factor and lactate dehydrogenase (LDH), both relevant in chronic inflammation. The von Hippel-Lindau protein (pVHL) is a negative regulator of HIF-1α. Previously, we found that pVHL was almost absent in the lesions of psoriasis patients; therefore, we investigated the impact of rescue pVHL expression in lesional skin. We used the imiquimod-induced psoriasis-like mouse model as an adenoviral vector that allowed us to express pVHL in the skin. Our data show that, in lesional skin, pVHL expression was reduced, whereas HIF-1α was increased. Remarkably, the retrieval of pVHL prevented psoriatic lesions, diminishing erythema, scale, and epidermal and vascular thickness. Furthermore, pVHL expression was capable of reducing HIF-1α, LDH, TNF-α and immune cell infiltration (mainly IL-17+ neutrophils). In conclusion, our results demonstrate that pVHL has a protective role to play in the pathophysiology of psoriasis.

IL-36 Cytokines: Regulators of Inflammatory Responses and Their Emerging Role in Immunology of Reproduction.

The IL-36 subfamily of cytokines has been recently described as part of the IL-1 superfamily. It comprises three pro-inflammatory agonists (IL-36α, IL-36ß, and IL-36γ), their receptor (IL-36R), and one antagonist (IL-36Ra). Although expressed in a variety of cells, the biological relevance of IL-36 cytokines is most evident in the communication between epithelial cells, dendritic cells, and neutrophils, which constitute the common triad responsible for the initiation, maintenance, and expansion of inflammation. The immunological role of IL-36 cytokines was initially described in studies of psoriasis, but novel evidence demonstrates their involvement in further immune and inflammatory processes in physiological and pathological situations. Preliminary studies have reported a dynamic expression of IL-36 cytokines in the female reproductive tract throughout the menstrual cycle, as well as their association with the production of immune mediators and cellular recruitment in the vaginal microenvironment contributing to host defense. In pregnancy, alteration of the placental IL-36 axis has been reported upon infection and pre-eclampsia suggesting its pivotal role in the regulation of maternal immune responses. In this review, we summarize current knowledge regarding the regulatory mechanisms and biological actions of IL-36 cytokines, their participation in different inflammatory conditions, and the emerging data on their potential role in normal and complicated pregnancies.

Peptidoglycan from Staphylococcus aureus has an anti-apoptotic effect in HaCaT keratinocytes mediated by the production of the cellular inhibitor of apoptosis protein-2.

Colonization of epithelium by microorganisms leads to inflammatory responses. In some cases an anti-apoptotic response involving the cellular inhibitor of apoptosis protein-2 (cIAP-2) also occurs. Although strong expression of cIAP-2 has been observed in lesional skin from psoriatic patients and in HaCaT keratinocytes treated with peptidoglycan (PGN) from Staphylococcus aureus, anti-apoptotic responses induced in the skin by cIAP-2 have seldom been studied. In this study, the effect of PGN on TNF-α-induced apoptotic HaCaT keratinocytes was assessed. Morphological analysis, quantification of cells with DNA fragmentation and active caspase-3 detection was performed to assess apoptotic cell death. Greater LL-37 and cIAP-2 production was found in keratinocytes stimulated with PGN than in non-treated cells (P < 0.05). In comparison with cells treated with TNF-α only, a significant reduction in apoptotic cell death was observed when HaCaT were pretreated with PGN before inducing apoptosis with TNF-α (P < 0.05). In addition, an inhibitor of cIAP-2 activity (LCL161) stopped the PGN effect. These findings show that PGN from S. aureus has an anti-apoptotic effect in keratinocytes mediated by cIAP-2 production, suggesting that this anti-apoptotic activity could favor proliferation of keratinocytes in psoriasis.

Non-biofilm-forming Staphylococcus epidermidis planktonic cell supernatant induces alterations in osteoblast biological function.

Staphylococcal biofilms significantly contribute to prosthetic joint infection (PJI). However, 40% of S. epidermidis PJI isolates do not produce biofilms, which does not explain the role of biofilms in these cases. We studied whether the supernatant from planktonic S. epidermidis alters osteoblast function. Non-biofilm-forming S. epidermidis supernatants (PJI- clinical isolate, healthy skin isolate (HS), and ATCC12228 reference strain) and biofilm-forming supernatants (PJI+ clinical isolate, ATCC35984 reference strain, and Staphylococcus aureus USA300 reference strain) were included. Osteoblasts stimulated with supernatants from non-biofilm-forming isolates for 3, 7, and 14 days showed significantly reduced cellular DNA content compared with unstimulated osteoblasts, and apoptosis was induced in these osteoblasts. Similar results were obtained for biofilm-forming isolates, but with a greater reduction in DNA content and higher apoptosis. Alkaline phosphatase activity and mineralization were significantly reduced in osteoblasts treated with supernatants from non-biofilm-forming isolates compared to the control at the same time points. However, the supernatants from biofilm-forming isolates had a greater effect than those from non-biofilm-forming isolates. A significant decrease in the expression of ATF4, RUNX2, ALP, SPARC, and BGLAP, and a significant increase in RANK-L expression were observed in osteoblasts treated with both supernatants. These results demonstrate that the supernatants of the S. epidermidis isolate from the PJI- and HS (commensal) with a non-biofilm-forming phenotype alter the function of osteoblasts (apoptosis induction, failure of cell differentiation, activation of osteoblasts, and induction of bone resorption), similar to biofilm-forming isolates (PJI+, ATCC35984, and S. aureus USA300), suggesting that biofilm status contributes to impaired osteoblast function and that the planktonic state can do so independently of biofilm production.

NF-κB and Its Regulators During Pregnancy.

The transcriptional factor NF-κB is a nuclear factor involved in both physiological and pathological processes. This factor can control the transcription of more than 400 genes, including cytokines, chemokines, and their modulators, immune and non-immune receptors, proteins involved in antigen presentation and cell adhesion, acute phase and stress response proteins, regulators of apoptosis, growth factors, other transcription factors and their regulators, as well as different enzymes; all these molecules control several biological processes. NF-κB is a tightly regulated molecule that has also been related to apoptosis, cell proliferation, inflammation, and the control of innate and adaptive immune responses during onset of labor, in which it has a crucial role; thus, early activation of this factor may have an adverse effect, by inducing premature termination of pregnancy, with bad outcomes for the mother and the fetus, including product loss. Reviews compiling the different activities of NF-κB have been reported. However, an update regarding NF-κB regulation during pregnancy is lacking. In this work, we aimed to describe the state of the art around NF-κB activity, its regulatory role in pregnancy, and the effect of its dysregulation due to invasion by pathogens like Trichomonas vaginalis and Toxoplasma gondii as examples.

IκBNS and IL-6 expression is differentially established in the uterus of pregnant healthy and infected mice.

During pregnancy, NF-κB plays an important role for embryo implantation and the onset of labor. Regulated IL-6 production, under transcriptional control of NF-κB, is essential for a successful pregnancy outcome and the atypical regulator IκBNS is involved in this process. Previously, we showed that IκBNS negatively regulates IL-6 in uterine tissues during mouse estrous cycle. In this work, we analyzed if IκBNS and IL-6 expression in pregnant mice under physiological or L. monocytogenes-infected conditions would remain as observed in estrous cycle. In the healthy pregnancy IL-6 was highly expressed during implantation/placentation and labor stages but decreased during fetal development and post-partum stages. In contrast, in mice infected before pregnancy, IL-6 expression was not increased in the implantation stage, and its regulator IκBNS increased more in the infected condition rather than in the healthy pregnancy. IκBNS expression was reduced in post-implantation infection, allowing for IL-6 overexpression. The IκBNS-unrelated cytokine IL-36γ, used as inflammatory cytokine marker, was severely increased in the infected uterine tissues. When we analyzed the effect of infection over the fetuses, we found that pre-implantation infection caused the resorption (rejection) of some products, while the post-implantation infection restricted the intrauterine growth of fetuses. The results suggest that in the uterine tissue of pregnant mice the regulatory effect of IκBNS over IL-6 is more evident in an infection status rather than in a healthy condition.

NRAMP1 Polymorphisms like Susceptibility Marker in Mexican Focus of Cutaneous Leishmaniasis.

Cutaneous leishmaniasis (CL) is endemic in Campeche state, Mexico. Host and parasite factors are involved in the establishment and development of CL. Host factors include immune response and genetic background. NRAMP1 (Natural Resistance Associated Macrophage Protein 1) is important in innate immunity. Polymorphisms in NRAMP1 have been associated with susceptibility or resistance to infectious and autoimmune diseases. To study the association of NRAMP1 mutations with CL in patients from Calakmul, Campeche, samples from 115 CL patients and 69 samples of healthy people from the same area were evaluated. Five regions in NRAMP1 were amplified and digested, looking for mutations in the promoter region (-524G/C), exon 3 (274C/T), exon 8 (823 C7T), and exon 15 (G/A) and deletion of 4 bp in the 3'UTR region. We found a statistical association between polymorphisms in 3'UTR region and exon 8 and CL [χ2 = 13.26; p < 0.05; OR = 17.00; IC of 95% (2.24-128.99)]. Some patients who needed more than 40 doses of Glucantime® to heal injuries presented mutations in exons 3, 8, and 15. Multiple or ear lesions were not associated with NRAMP1 polymorphism.

Estrous Cycle and Gestational Age-Dependent Expression of Members of the Interleukin-36 Subfamily in a Semi-Allogeneic Model of Infected and Non-Infected Murine Pregnancy.

The IL-36 subfamily is a recently described group of cytokines with pro-inflammatory behavior, comprising three agonists (α, ß, and γ), its receptor (R), and one antagonist (Ra). The expression and function of IL-36 subfamily members in the estrous cycle in healthy and infected pregnancy has not been described. We evaluated mRNA and protein expression of IL-36 family members during the estrous cycle, implantation, fetal development, and post-labor periods in a model of allogenic pregnancy in mice. We also explored the ability of Listeria monocytogenes to modulate the expression of IL-36 subfamily members during pregnancy. Expression of IL-36 subfamily members showed different expression during the estrous cycle and pregnancy but was induced at estrous, 16.5 days post coitum (dpc), 18.5 dpc, and labor. IL-36 subfamily members showed a characteristic distribution in the glandular epithelium, perimetrium, myometrium, and stratum vasculare. Infection with L. monocytogenes during pregnancy induced strong production of IL-36 subfamily members, an observation that correlated with an increasing prevalence of fetal loss. In conclusion, IL-36 agonists showed specific patterns of mRNA and protein expression that might suggest functional specialization or specific target cells. Infection with L. monocytogenes during pregnancy induced strong production of IL-36 subfamily members.

CD4 and CD8 T cell response to the rHSP60 from Klebsiella pneumoniae in peripheral blood mononuclear cells from patients with ankylosing spondylitis.

OBJECTIVE: To determine the processing pathways used by peripheral blood mononuclear cells (PBMC) and present the rHSP60Kp, and the T cell subpopulations involved in the response, in patients with ankylosing spondylitis (AS) METHODS: The lymphoproliferative response to the rHSP60Kp in PBMC from 14 HLA-B27+ AS patients and 15 B27- healthy controls was assessed by 3H-TdR incorporation. The processing pathways for the rHSP60Kp were analyzed by 3H-TdR incorporation in fresh PBMC from patients using homologous PBMC preincubated with the antigen and specific inhibitors: chloroquine, N-acetyl-L-leucil-L-leucil-L-nor-leucinal (LLnL) or brefeldin A (BFA), fixed with p-formaldehyde (fixed APC). The CD4+/CD8+ T cell subpopulation activated with the antigen was determined by three colours flow cytometry in PBMC from patients. RESULTS: Eight out of fourteen patients showed positive lymphoproliferative responses to the rHSP60Kp while none of the healthy controls responded (p < 0.012). In five patients S.I. was above 4.0. In these patients lymphoproliferation was lower when chloroquine and LLnL was used and it became negative with BFA, indicating that both pathways are used. CD4+ and CD8+ T cells populations expressed CD69 when activated by the rHSP60Kp. CONCLUSIONS: Our results suggest that CD4 and CD8 T cells participate in the response to the rHSP60Kp in B27+ AS patients.

LL-37 regulates the overexpression of vascular endothelial growth factor (VEGF) and c-IAP-2 in human keratinocytes.

BACKGROUND: The antimicrobial peptide PR39 is a porcine cathelicidin with angiogenic and antiapoptotic activities, as it can regulate the expression of vascular endothelial growth factor (VEGF) and inhibitor apoptosis protein-2 (c-IAP-2) in endothelial cells. The human homolog LL-37 has been found to be highly expressed in human keratinocytes from psoriatic patients, but it is not known whether LL-37 can modulate the expression of VEGF and c-IAP-2 in keratinocytes, as both molecules are involved in the overgrowth of psoriatic skin. Therefore, in this work, we studied the possible role of CAP18/LL-37 in the modulation of VEGF and c-IAP-2 expression in human keratinocytes. METHODS: The CAP18/LL-37 gene was cloned into a plasmid that contained green fluorescent protein (GFP). This plasmid was called pGFP-CAP18/LL-37. The expression of LL-37, VEGF, and c-IAP-2 was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting in HaCaT cells transfected with pGFP-CAP18/LL-37. Specific DNAzymes were used to break the CAP18/LL-37 mRNA (DNAz-CAP18/LL-37). RESULTS: HaCaT cells transfected with pGFP-CAP18/LL-37 showed the upregulation of VEGF and c-IAP-2 mRNAs. Hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA expression did not change during the assays; however, its protein was increased, as well as the VEGF protein. HaCaT cells cotransfected with pGFP-CAP18/LL-37 and DNAz-CAP18/LL-37 showed depleted expression of LL-37, VEGF, and c-IAP-2 mRNAs. CONCLUSIONS: These results suggest that LL-37 may modulate the expression of VEGF and c-IAP-2 via HIF-1alpha in human keratinocytes.

Isolation, vancomycin resistance and biofilm production of Staphylococcus epidermidis from patients with conjunctivitis, corneal ulcers, and endophthalmitis.

The infection frequency associated to bacterial conjunctivitis, corneal ulcers (CU), and endophthalmitis was studied along a five years period. The isolation and identification of microorganisms were performed by culture-based methods and biochemical test respectively. Also, a nested PCR to detect gram-negative and gram-positive bacteria in the clinical samples was assayed. Nested PCR was a more efficient method than culture to detect bacteria in the samples. The most frequently isolated species was Staphylococcus epidermidis, a bacterium commonly considered as a human saprophyte. The S. epidermidis strains from conjunctivitis, CU, and endophthalmitis exhibited 46, 33.9, and 34.1% of oxacilin-resistance respectively. A total of 28% of intermediate-vancomycin resistance (MIC = 8-16 microg/ml) was observed among S. epidermidis strain collection. The UPGMA cluster analysis of the multiresistance profile data of intermediate vancomycin-resistant S. epidermidis strains showed a high phenotypic diversity and no relationship between each group and their clinical origin. The biofilm formation capacity was broadly distributed (66%), particularly among intermediate-vancomycin strains (> 75%). In brief, S. epidermidis displayed a high diversity of antibiotic resistance profiles and biofilm formation capacity. These phenotypic traits could explain the high isolation frequency of S. epidermidis from ocular infections and oblige to review the saprophytic status of these bacteria.

CD4 and CD8 T cell response to the rHSP60 from Klebsiella pneumoniae in peripheral blood mononuclear cells from patients with ankylosing spondylitis / Respuesta de linfocitos T CD4 y CD8 contra la rHSP60 de Klebsiella pneumoniae en células mononucleares de sangre periférica de pacientes con espondilitis anquilosa

Objective. To determine the processing pathways used by peripheral blood mononuclear cells (PBMC) and present the rHSP60Kp, and the T cell subpopulations involved in the response, in patients with ankylosing spondylitis (AS) Methods. The lymphoproliferative response to the rHSP60Kp in PBMC from 14 HLA-B27 + AS patients and 15 B27 healthy controls was assessed by ³H-TdR incorporation. The processing pathways for the rHSP60Kp were analyzed by ³H-TdR incorporation in fresh PBMC from patients using homologous PBMC preincubated with the antigen and specific inhibitors: chloroquine, N-acetyl-L-leucil-L-leucil-L-nor-leucinal (LLnL) or brefeldin A (BFA), fixed with p-formaldehyde (fixed APC). The CD4+/CD8+ T cell subpopulation activated with the antigen was determined by three colours flow cytometry in PBMC from patients. Results. Eight out of fourteen patients showed positive lymphoproliferative responses to the rHSP60Kp while none of the healthy controls responded (p < 0.012). In five patients S.I. was above 4.0. In these patients lymphoproliferation was lower when chloroquine and LLnL was used and it became negative with BFA, indicating that both pathways are used. CD4+ and CD8+ T cells populations expressed CD69 when activated by the rHSP60Kp. Conclusions. Our results suggest that CD4 and CD8 T cells participate in the response to the rHSP60Kp in B27+ AS patients.

Objetivo.Determinar las vías utilizadas por las células mononucleares de sangre periférica (CMSP) de pacientes con espondilitis anquilosante para procesar a la rHSPGO de Klebsiella pneumoniae (rHSPGOKp) y las subpoblaciones de linfocitos T involucrados en la activación. Métodos. Se determinó la respuesta linfoproliferativa, por incorporación de ³H-TdR en CMSP, en presencia de la rHSPGOKp, en 14 pacientes con EA HLA-B27+y en 15 sujetos sanos HLA-B27-. La ruta de procesamiento y presentación de la rHSPGOKp se determinó por incorporación de ³H-TdR en las CMSP de los pacientes utilizando como células presentadoras a las CMSP homologas, preíncubadas con el antígeno y los inhibidores específicos: cloroquína, brefeldína A y N-acetil-L-leucil-L-leucil-L-nor-leucinal (LLnL), y fijadas con p-formaldehído. Se evaluaron las subpoblaciones de linfocitos T CD4+ y CD8+ que expresaron CD69, frente al antígeno, por citometría de flujo. Resultados. Ocho de los 14 pacientes y ninguno de los sujetos sanos, tuvo respuesta linfoproliferativa positiva (IE > 3.0) contra la rHSPGOKp (p < 0.012). En cinco de los pacientes el I.E. fue superior a 4.0. En estos pacientes la linfoproliferación disminuyó cuando se utilizó cloroquína y LLnL, y se hizo negativa cuando se utilizó BFA, lo que indica que ambas vías son empleadas. Las subpoblaciones de linfocitos T (CD4+ y CD8+) expresaron CD69 frente al antigeno. Conclusiones. Nuestros resultados sugieren que ambas poblaciones de linfocitos T: CD4+ y CD8+ participan en la respuesta a la rHSPGOKp.