Alena Sekerkova

Cytokine networking of innate immunity cells: a potential target of therapy

Innate immune cells, particularly macrophages and epithelial cells, play a key role in multiple layers of immune responses. Alarmins and pro-inflammatory cytokines from the IL (interleukin)-1 and TNF (tumour necrosis factor) families initiate the cascade of events by inducing chemokine release from bystander cells and by the up-regulation of adhesion molecules required for transendothelial trafficking of immune cells. Furthermore, innate cytokines produced by dendritic cells, macrophages, epithelial cells and innate lymphoid cells seem to play a critical role in polarization of helper T-cell cytokine profiles into specific subsets of Th1/Th2/Th17 effector cells or regulatory T-cells. Lastly, the innate immune system down-regulates effector mechanisms and restores homoeostasis in injured tissue via cytokines from the IL-10 and TGF (transforming growth factor) families mainly released from macrophages, preferentially the M2 subset, which have a capacity to induce regulatory T-cells, inhibit the production of pro-inflammatory cytokines and induce healing of the tissue by regulating extracellular matrix protein deposition and angiogenesis. Cytokines produced by innate immune cells represent an attractive target for therapeutic intervention, and multiple molecules are currently being tested clinically in patients with inflammatory bowel disease, rheumatoid arthritis, systemic diseases, autoinflammatory syndromes, fibrosing processes or malignancies. In addition to the already widely used blockers of TNFα and the tested inhibitors of IL-1 and IL-6, multiple therapeutic molecules are currently in clinical trials targeting TNF-related molecules [APRIL (a proliferation-inducing ligand) and BAFF (B-cell-activating factor belonging to the TNF family)], chemokine receptors, IL-17, TGFβ and other cytokines.

B-cell-related biomarkers of tolerance are up-regulated in rejection-free kidney transplant recipients.

Background Molecular signatures have recently been identified in operationally tolerant long-term kidney transplant patients; however, their expression in patients on immunosuppression remains unclear. Methods In this prospective study, the gene expression profiles of eight selected tolerance-associated genes (MS4A1, CD79B, TCL1A, TMEM176B, FOXP3, TOAG-1, MAN1A1, and TLR5) in the peripheral blood of 67 kidney transplant recipients at days 0, 7, 14, 21, 28, 60, 90, and at 6 and 12 months, and in graft biopsies were measured. Similarly, using flow cytometry, CD45+CD19+CD3− B-cell counts were evaluated in the follow-up. Expression patterns were compared among patients with biopsy-proven acute rejection, borderline changes, and in rejection-free patients. A generalized linear mixed model with gamma distribution for repeated measures adjusted for induction therapy was used for statistical analysis of longitudinal data and Kruskal-Wallis test for case biopsy data. Results Compared to patients with rejection, a significantly higher number of peripheral B cells were observed during follow-up in rejection-free patients and in patients with borderline changes. Gene expression patterns of MS4A1 (CD20), TCL1A, CD79B, TOAG-1, and FOXP3 genes were significantly higher in rejection-free patients as compared to rejection group with the highest differences during the first 3 months. In contrast, TMEM176B (TORID) was up-regulated in the rejection group. Similar trends were also observed between patients with borderline changes and acute rejection. Higher intragraft expression of TOAG-1 was observed in rejection-free patients. Conclusions These observations suggest an association of B-cell signatures, seen also in drug-free tolerant patients, with controlled alloimmune response.

Detection of Phl p 1, Phl p 5, Phl p 7 and Phl p 12 Specific IgE Antibodies in the Sera of Children and Adult Patients Allergic to Phleum Pollen

BACKGROUND Grasses belong to major sources of inhaled allergens. The knowledge of particular molecules responsible for hypersensitivity is of crucial importance for better understanding of individual differences among single allergic subjects and allergic populations living in various world-areas. METHODS Specific-IgE-antibodies against Phl p 1, Phl p 5, Phl p 7, Phl p 12 were detected in a group of 130 Phleum-allergic-subjects (82 children, 48 adults). RESULTS Phl p 1 antibodies were detected in most pediatric and adult patients, however, the children were associated with higher RAST classes more often. Anti-Phl p 5-antibodies were found more frequently in adults. An increase was observed in the number of pediatric patients reacting to Phl p 7 and Phl p 12. There were no differences in concentrations of specific-IgE against Phl p 5, Phl p 7 and Phl p 12 depending on age. Almost 10% of allergic children produced antibodies directed exclusively against minor allergens or did not produce specific-IgE-antibodies against tested molecules. Part of the patients reacted to profilin and calcium-binding protein originating from only one source (Phl p 12/Bet v 2 and Phl p 7/Bet v 4). CONCLUSIONS Antibodies against Phl p 1 and Phl p 5 can be used as a marker of allergy to grasses in adult patients. Children reacted exclusively to minor allergens more frequently than adults. Prolonged allergen exposure is evidently necessary to induce sensitization to Phl p 5. A high level of homology between profilins and calcium-binding proteins enables only one allergen to be used for diagnostic purposes but a possibility of a reaction to species-bound epitopes should be taken into account.

CD14+CD16+ and CD14+CD163+ monocyte subpopulations in kidney allograft transplantation

BackgroundMonocytes represent a heterogeneous population of cells subdivided according to the expression level of membrane antigens. A pro-inflammatory (intermediate/nonclassical) subpopulation of monocytes is defined by expression of CD16. CD163 seems to be characteristically preferentially expressed by immunosuppressive monocytes. The aim of our study was to evaluate the distribution of monocyte subpopulations in 71 patients with kidney allograft transplantation.ResultsThe phenotype was evaluated by flow cytometry in defined time points. The proportions of peripheral CD14+CD16+ monocytes were downregulated immediately after the kidney transplantation and basiliximab treatment partially attenuated this trend. The transient downregulation of the CD14+CD16+ subpopulation was adjusted to basal values in two months. The proportions of CD14+CD163+ monocytes were transiently upregulated early after the kidney transplantation and remained higher during the first month in most patients. In ATG treated patients, the expansion of CD14+CD163+ monocytes was delayed but their upregulation lasted longer. In vitro data showed the direct effect of ATG and methylprednisolone on expression of CD16 and CD163 molecules while basiliximab did not affect the phenotype of cultured monocytes.ConclusionsWe assume from our data that kidney allograft transplantation is associated with modulation of monocyte subpopulations (CD14+CD16+ and CD14+CD163+) partially affected by an immunosuppressive regime used.

Dynamic changes of B‐cell compartments in kidney transplantation: lack of transitional B cells is associated with allograft rejection

B cells play an important role in the immune responses which affect the outcomes of kidney allografts. Dynamic changes of B‐cell compartments in clinical kidney transplantation are still poorly understood. B‐cell subsets were prospectively monitored using flow cytometry for 1 year in 98 kidney transplant recipients. Data were correlated with immunosuppression and clinical outcomes. An increase in the total population of B lymphocytes was observed during the first week after transplantation. The level of IgMhighCD38highCD24high transitional B cells reduced significantly up until the third month, with partial repopulation in the first year. Lower numbers of transitional B cells in the third month were associated with higher risk of graft rejection. IgM+IgD+CD27− naive B cells did not change within follow‐up. IgM+CD27+ nonswitched memory B cells and IgM−CD27+ switched memory B cells increased on post‐operative day 7. IgM−CD38highCD27high plasmablasts showed similar kinetics during the first post‐transplant year, similar to transitional B cells. In conclusion, sensitized kidney transplant recipients as well as those with either acute or chronic rejection within the first post‐transplant year exhibited lower levels of transitional B cells. Therefore, these data further support the hypothesis that transitional B cells have a protective role in kidney transplantation.

Effect of induction therapy on the expression of molecular markers associated with rejection and tolerance

BackgroundInduction therapy can improve kidney transplantation (KTx) outcomes, but little is known about the mechanisms underlying its effects.MethodsThe mRNA levels of T cell-related genes associated with tolerance or rejection (CD247, GZMB, PRF1, FOXP3, MAN1A1, TCAIM, and TLR5) and lymphocyte subpopulations were monitored prospectively in the peripheral blood of 60 kidney transplant recipients before and 7, 14, 21, 28, 60, 90 days, 6 months, and 12 months after KTx. Patients were treated with calcineurin inhibitor-based triple immunosuppression and induction with rabbit anti-thymocyte globulin (rATG, n = 24), basiliximab (n = 17), or without induction (no-induction, n = 19). A generalized linear mixed model with gamma distribution for repeated measures, adjusted for rejection, recipient/donor age and delayed graft function, was used for statistical analysis.ResultsrATG treatment caused an intense reduction in all T cell type population and natural killer (NK) cells within 7 days, then a slow increase and repopulation was observed. This was also noticed in the expression levels of CD247, FOXP3, GZMB, and PRF1. The basiliximab group exhibited higher CD247, GZMB, FOXP3 and TCAIM mRNA levels and regulatory T cell (Treg) counts than the no-induction group. The levels of MAN1A1 and TLR5 mRNA expressions were increased, whereas TCAIM decreased in the rATG group as compared with those in the no-induction group.ConclusionThe rATG induction therapy was associated with decreased T and NK cell-related transcript levels and with upregulation of two rejection-associated transcripts (MAN1A1 and TLR5) shortly after KTx. Basiliximab treatment was associated with increased absolute number of Treg cells, and increased level of FOXP3 and TCAIM expression.

Steroid free immunosuppression is associated with enhanced Th1 transcripts in kidney transplantation

BACKGROUND Steroid avoidance in immunosuppression in kidney transplantation offers several metabolic advantages, however it is associated with higher early acute rejection rate. Cellular and molecular mechanisms of this phenomenon remain poorly understood. METHODS In this single center observational study, low-risk kidney transplant recipients randomized into large multicenter prospective ADVANCE trial with steroid avoidance/early withdrawal and center standard of care treated patients were monitored for 12months. The expressions of 28 transcripts, associated with alloimmune response and operational tolerance, were evaluated in the peripheral blood using RT-qPCR at 0, 7, 14, 90 and 365 postoperative days (POD) and in the protocol graft biopsy at 3months while lymphocyte subpopulations were analyzed by flow-cytometry within the follow-up. RESULTS Both steroid avoidance and withdrawal regimens were associated with significantly higher granzyme B (GZMB) transcript at POD 14 and perforin 1 (PRF1) transcript at POD 7. The higher interleukin 2 (IL-2) expression at POD 7 was detected only in the steroid avoidance group. Initial steroids decreased the expression SH2D1B transcript at POD14 and there were no further differences in other operational tolerance transcripts among groups. The statistically significant decrease in absolute numbers of peripheral NK cells in the first 14days was observed in the standard of care group only. There were no differences in analyzed intrarenal transcripts in 3-month biopsies among groups. CONCLUSIONS The enhanced expression of some of Th1 associated transcripts and limited effects on NK cells of steroid avoidance immunosuppression suggest higher susceptibility for early acute rejection.

Manumycin A downregulates release of proinflammatory cytokines from TNF alpha stimulated human monocytes

Macrolide antibiotics such as azithromycin or clarithromycin are known to have potent anti-inflammatory and immunomodulatory effects but these properties cannot be widely used due to a risk of bacterial resistance. We studied another polyketide antibiotic, structurally related manumycin A known as a streptomycete derived farnesyltransferase inhibitor with limited antibacterial effects, with respect to its potential regulation of mRNA expression of several genes associated with proinflammatory responses. Downregulation of mRNA for IL-6, TLR-8, IL-1 beta and IL-10 was found in THP-1 cells after 4h stimulation with TNF alpha in the presence of manumycin A and downregulated TLR-8 and EGR-1 genes were observed after 8h. Among the genes upregulated in response to manumycin were HMOX-1, TNFRSF10A, IL-1R1, TICAM2, NLRP12 after 4h and only IL-1R1 after 8h. Furthermore, manumycin A was found to inhibit IL-1beta, IL-6, and IL-8 production in TNF alpha stimulated THP-1 cells and peripheral blood monocytes in a dose dependent manner (0.25-1 μM of manumycin A) without affecting cell viability. Cell viability of blood monocytes decreased by about 30% at manumycin A doses of 2-5 μM. Manumycin A also inhibited IL-18 release from THP-1 cells, while in cultures of blood monocytes, this cytokine was not detectable. That manumycin A mediated downregulation of proinflammatory genes in human monocytes confirmed by a measurement of cytokine levels in culture supernatants, together with a very limited effect on cell viability, might suggest potential anti-inflammatory properties of this polyketide antibiotic.

Biphasic response in number of stem cells and endothelial progenitor cells after left ventricular assist device implantation: A 6 month follow-up ☆

BACKGROUND Continuous blood flow could have deleterious effects on endothelium and vascular health. This could have serious consequences in patients with heart failure treated with continuous flow left ventricular assist devices (LVAD). Therefore, we studied effect of LVAD on three circulating vascular biomarkers: stem cells (SC), endothelial progenitor cells (EPC) and microparticles (MP). METHODS In 23 patients (5 women) with end-stage heart failure, SC, EPC and MP were measured before, and 3 and 6months after implantation of LVAD (HeartMate II). SC were defined using determination of surface antigen expression as mononuclear CD34+/CD45low+ cells and EPC as mononuclear CD34+/CD45low+/KDR+ cells. MP concentrations were determined by ELISA method. RESULTS Three months after LVAD implantation numbers of SC and EPC significantly decreased (p=0.01 and p=0.001, respectively). On the contrary, between 3rd and 6th month after implantation they significantly increased (p=0.006 and p=0.003, respectively).MP did not change significantly during the study despite exerting similar trend as SC and EPC. CONCLUSIONS Observed biphasic changes of SC and EPC might reflect two processes. First, shortly after LVAD implantation, improved tissue perfusion could lead to decrease in ischemic stimuli and ensuing decrease of SC and EPC. Second, continuous flow between 3rd and 6th month produced by LVAD could lead to increase of SC and EPC through activation of endothelium. This explanation could be supported also by similar trend in the changes of concentrations of MP.

High Prevalence of Neutrophil Cytoplasmic Autoantibodies in Infants with Food Protein-Induced Proctitis/Proctocolitis: Autoimmunity Involvement?

Background. Food protein-induced proctitis/proctocolitis (FPIP) is the most common noninfectious colitis in children in the first year of life. Along with the overall clinical symptoms, diarrhoea and rectal bleeding are the main manifestations of the disease. There is no routine noninvasive test that would be specific for this type of colitis. The aim of our study was to find a noninvasive laboratory test or tests that may be helpful in differential diagnosis of food protein-induced proctitis/proctocolitis. Methods. ANA, ANCA, ASCA, a-EMA, a-tTg, specific IgE, total IgE, IgG, IgA, IgM, and concentration of serum calprotectin were measured in a group of 25 patients with colitis and 18 children with other diagnoses. Results. Atypical-pANCA antibodies of IgG isotype were detected in the sera of 24 patients by the method of indirect immunofluorescence, and 5 patients showed also the positivity of IgA isotype. In control samples these autoantibodies were not detected. Other autoantibodies were not demonstrated in either patient or control group. Conclusions. Of the parameters tested in noninfectious colitis, atypical-pANCA on ethanol-fixed granulocytes appears to be a suitable serological marker of food protein-induced proctitis/proctocolitis and suggests a possible involvement of an autoimmune mechanisms in the pathogenesis of this disease.