Complement receptors 3 and 4 in kidney diseases

CR3 and CR4 are among the most abundant molecules on the cell surface of DCs, monocytes/macrophages, and PMNs. Both integrins recognize common ligands among which are iC3b, ICAM-1, and fibrinogen. iC3b is generated as a result of the activation of the complement cascade and can opsonize antigens. Among the diseases characterized by the activation of the complement system are glomerulonephritis and pyelonephritis. This project aimed to investigate the influence of CR3 and CR4 on the progression of these diseases using our newly generated ItgamItgax-/- mouse line. During the project, we found that genes encoding CR3 and CR4 are upregulated in patients suffering from several types of glomerulonephritis. Also, mice lacking both CR3 and CR4 showed attenuated symptoms of the disease, as evidenced by lower ACR and concentration of NGAL in their urine compared to WT animals. Histological analysis of kidneys from ItgamItgax-/- mice showed a lower degree of crescent formation. Flow cytometric analysis revealed a decrease in the number of cDC2s and CD4+ T cells in the kidneys of ItgamItgax-/- mice compared to the WT group, which can indicate a selective decrease in inflammation. CD4+ T cells of nephritic ItgamItgax-/- mice had also a lower expression of genes engaged in IL-1-related signaling. cDC2s of ItgamItgax-/- mice captured less antigen during cGN compared to WT cells. Also, they were less efficient at inducing the proliferation of CD4+ T cells compared to WT DCs. In the case of pyelonephritis, ItgamItgax-/- animals had also attenuated symptoms of the disease as evidenced by the lower bacterial load in their kidneys compared to the WT group and decreased inflammation on day 7 p.i. ItgamItgax-/- BMDMs contained also less viable intracellular bacteria compared to WT cells, which can indicate that due to the absence of CR3 and CR4 bacteria are not efficiently phagocytose. This led us to the conclusion that UPEC can use macrophages as a niche to evade immune response in WT animals, which is not possible in the case of ItgamItgax-/- mice. This can explain the better outcome of knockout animals. The findings presented above reveal the importance of CR3 and CR4 during the progression of crescentic glomerulonephritis as well as pyelonephritis and suggest that targeting these receptors may be a therapeutic strategy in the treatment of these diseases

Inactive Gingipains from P. gingivalis Selectively Skews T Cells toward a Th17 Phenotype in an IL-6 Dependent Manner

Gingipain cysteine proteases are considered key virulence factors of Porphyromonas gingivalis. They significantly influence antibacterial and homeostatic functions of macrophages, neutrophils, the complement system, and cytokine networks. Recent data indicate the role of P. gingivalis in T cell differentiation;however, the involvement of gingipains in this process remains elusive. Therefore, the aim of this study was to investigate the contribution of danger signals triggered by the gingipains on the generation of Th17 cells, which play a key role in protection against bacterial diseases but may cause chronic inflammation and bone resorption. To this end we compared the effects of the wild-type strain of P. gingivalis (W83) with its isogenic mutant devoid of gingipain activity (Delta K Delta RAB), and bacterial cells pretreated with a highly-specific inhibitor of gingipains activity (KYTs). Antigen presenting cells (APCs), both professional (dendritic cells), and non-professional (gingival keratinocytes), exposed to viable bacteria expressed high amounts of cytokines (IL-6, IL-21, IL-23). These cytokines are reported to either stimulate or balance the Th17-dependent immune response. Surprisingly, cells infected with P. gingivalis devoid of gingipain activity showed increased levels of all tested cytokines compared to bacteria with fully active enzymes. The effect was dependent on both the reduction of cytokine proteolysis and the lack of cross-talk with other bacterial virulence factors, including LPS and fimbriae that induce de novo synthesis of cytokines. The profile of lymphocyte T differentiation from naive T cells showed enhanced generation of Th17 in response to bacteria with inactive gingipains. Moreover, we found that gingipain-dependent induction of Th17 cells was highly specific, since other T cell-subsets remained unchanged. Finally, inhibition of IL-6 signaling in dendritic cells led to a significant depletion of the Th17 population. Cumulatively, this study revealed a previously undisclosed role of gingipain activity in the process of Th17 differentiation reliant on blocking signaling through IL-6. Since inactivation of gingipains accelerates the skewing of T cells toward Th17 cells, which are detrimental in periodontitis, IL-6 signaling may serve as an attractive target for treatment of the disease

The analysis of anti-iflammatory role of conjugate of enkephalin and temporin peptides

Peptydy terapeutyczne są nową klasą leków, które mogą być stosowane w leczeniu licznych schorzeń, takich jak: choroby nowotworowe, metaboliczne, autoimmunizacyjne oraz zakaźne. Peptydy przeciwdrobnoustrojowe (ang. antimicrobial peptides, AMPs) są podgrupą peptydów terapeutycznych działających w zakażeniach bakteryjnych w sposób bezpośredni i pośredni. Wpływają one na eliminację mikroorganizmów oddziałując z ich ścianą lub błoną komórkową. Ponadto, są zdolne do neutralizacji bakteryjnych czynników zjadliwości oraz do modulacji odpowiedzi immunologicznej gospodarza. Jedną z najbardziej atrakcyjnych cech wykorzystania AMPs w medycynie jest niskie prawdopodobieństwo rozwoju oporności bakterii na wyżej wymienione związki.Celem niniejszej pracy była ocena właściwości przeciwzapalnych peptydów będących koniugatami syntetycznych analogów temporyny 1-CEb (DK5) i ludzkiej enkefaliny (DAL). W pracy podjęto także próbę wyjaśnienia mechanizmu antyzapalnego działania tych koniugatów.Podczas badań wykorzystano dwa modele komórkowe: model makrofagów ludzkich (ang. human monocyte-derived macrophages, HMDMs) i model linii komórkowej makrofagów mysich RAW264.7. W celu zbadania wpływu peptydów na odpowiedź zapalną wywołaną endotoksyną bakteryjną zbadano poziom ekspresji następujących cytokin i chemokin: TNFα, IL-6, IL-8 oraz IL-10. Ponadto, oceniono indukcję tlenku azotu, jako wskaźnika prozapalnej aktywacji makrofagów. Na podstawie uzyskanych wyników wykazano, że spośród testowanych peptydów koniugat DAL-PEG-DK5 wykazuje najsilniejsze działanie antyzapalne. Za pomocą cytometrii przepływowej dowiedziono, że antyzapalne działanie koniugatu DAL-PEG-DK5 wynika z ograniczenia wiązania LPS do błony komórkowej makrofagów. Ostatecznie udowodniono, że koniugat peptydów pod wpływem LPS ulega wzmożonej agregacji, co ma negatywny wpływ na rozpoznanie amyloidowych struktur endotoksyny przez receptory błony komórkowej gospodarza.Therapeutic peptides are a novel class of drugs, which can be used in treatment of various diseases, e.g. cancer, metabolic, autoimmune and infectious diseases. Antimicrobial peptides (AMPs) are a group of therapeutic compounds that can play an important role during the resolution of infection. The most common mechanism of action of AMPs relies on their ability to interact with bacterial cell walls or membranes. Moreover, AMPs can neutralize virulence factors of pathogens and modulate host immune response. One of the most attractive features of AMPs as potential therapeutics is a low possibility of the development of resistance towards AMPs by bacteria.The aim of the research was to evaluate anti-inflammatory properties of the peptide conjugate DAL-PEG-DK5 and investigate its mechanism of action. This peptide conjugate was generated by linkage of synthetic antimicrobial peptide DK5 (1-CEb temporin analogue) and synthetic analogue of human enkephalin (DAL).During the research two in vitro models were used: human monocyte derived macrophages (HMDMs) and murine macrophages cell line RAW264.7. In order to investigate the influence of the peptide conjugate on inflammation the level of expression of certain cytokines and chemokines (TNFα, IL-6, IL-8 and IL-10) was measured. Induction of nitric oxide, which is one of the main inflammatory mediators, was also evaluated. Obtained data showed that among tested peptides the most efficient anti-inflammatory activity was executed by DAL-PEG-DK5 conjugate. Flow cytometry experiments revealed that the anti-inflammatory mechanism of DAL-PEG-DK5 results from its ability to restrict binding of LPS to cell surface of macrophages. Finally, the results obtained during the research show that tested peptide conjugate aggregates in the presence of LPS, which results in impaired ability of the cell surface receptors to recognize amyloid structures of endotoxin

Influence of gingipain RgpA and its proteolytic activity on LPS- and FimA-induced IL-6 expression

Gingipainy są to proteazy cysteinowe produkowane przez bakterię Porphyromonas gingivalis. Gingipainy są uważane są za główne czynniki zjadliwości P. gingivalis, bowiem zostały one zidentyfikowane we wszystkich szczepach danej bakterii wyizolowanych od pacjentów cierpiących na paradontozę. Ponadto, wykazano, że poziom ekspresji gingipain koreluje z zaostrzeniem klinicznych parametrów choroby. Gingipainy mają duży wpływ na odpowiedź swoistą i nieswoistą, np. są one zdolne do proteolizy receptora CD14 obecnego na monocytach oraz wykazano zdolność danych enzymów do proteolitycznej degradacji IL-12 i IL-6. Jednakże wiedza na temat pośredniej roli tych enzymów w procesie modulacji ścieżek sygnałowych uruchamianych w wyniku działania innych czynników wirulencji P. gingivalis, tj. LPS oraz FimA nadal pozostaje skromna, zaś wpływ nieaktywnych gingipain na szlaki sygnałowe uruchamiane w odpowiedzi na wspomniane czynniki wirulencji pozostaje nieznany.Celem przedstawionej pracy była kompleksowa ocena wpływu nieaktywnego i aktywnego enzymu RgpA, LPS i fimbrii na ekspresję genu kodującego IL-6 w ludzkich makrofagach (HMDMs) i komórkach dendrytycznych (moDCs) orza ocena zjawiska cross-talk, czyli wzajemnego wpływu ścieżek przekazu sygnału indukowanych wyżej wymienionymi czynnikami wirulencji na ekspresję IL-6.Gingipains are cysteine proteinases produced by Porphyromonas gingivalis. Gingipains are considered to be the main virulence factors of P. gingivalis. They were identified in all clinical isolates, and the expression level of these enzymes correlates with exacerbation of periodontitis. Gingipains have a huge influence on adaptive and innate immune response, e.g. they are able to degrade monocyte CD14. Also it was shown that these enzymes are able to degrade IL-12 and IL-6. However, it is still unclear what is the role of gingipains in modulation of LPS- or FimA-induced signal transduction pathways. Moreover, there is no data about the influence of inactive gingipains on signal transduction pathways, which are induced by other virulence factors of P. gingivalis, e.g. LPS and FimA.The purpose of this study was to evaluate the influence of active and inactive enzyme RgpA, LPS and FimA on IL-6 gene expression in human monocyte-derived macrophages (HMDMs) and human monocyte-derived dendritic cells (moDCs) and to evaluate occurrence of cross-talk, i.e. the mutual influence of signal transduction pathways induced by active and inactive RgpA, LPS and FimA on IL-6 expression

A high-salt diet compromises antibacterial neutrophil responses through hormonal perturbation

The Western diet is rich in salt, which poses various health risks. A high-salt diet (HSD) can stimulate immunity through the nuclear factor of activated T cells 5 (Nfat5)-signaling pathway, especially in the skin, where sodium is stored. The kidney medulla also accumulates sodium to build an osmotic gradient for water conservation. Here, we studied the effect of an HSD on the immune defense against uropathogenic E. coli-induced pyelonephritis, the most common kidney infection. Unexpectedly, pyelonephritis was aggravated in mice on an HSD by two mechanisms. First, on an HSD, sodium must be excreted; therefore, the kidney used urea instead to build the osmotic gradient. However, in contrast to sodium, urea suppressed the antibacterial functionality of neutrophils, the principal immune effectors against pyelonephritis. Second, the body excretes sodium by lowering mineralocorticoid production via suppressing aldosterone synthase. This caused an accumulation of aldosterone precursors with glucocorticoid functionality, which abolished the diurnal adrenocorticotropic hormone-driven glucocorticoid rhythm and compromised neutrophil development and antibacterial functionality systemically. Consistently, under an HSD, systemic Listeria monocytogenes infection was also aggravated in a glucocorticoid-dependent manner. Glucocorticoids directly induced Nfat5 expression, but pharmacological normalization of renal Nfat5 expression failed to restore the antibacterial defense. Last, healthy humans consuming an HSD for 1 week showed hyperglucocorticoidism and impaired antibacterial neutrophil function. In summary, an HSD suppresses intrarenal neutrophils Nfat5-independently by altering the local microenvironment and systemically by glucocorticoid-mediated immunosuppression. These findings argue against high-salt consumption during bacterial infections