Indukcija i reindukcija eksperimentalnog alergijskog encefalomijelitisa u DA pacova - uticaj starosti i encefalitogena

Susceptibility to experimental allergic encephalomyelitis (EAE) was investigated in DA rats of both sexes, aged 5, 8 or 27 weeks. Guinea pig or Lewis rat spinal cords (GPSC or RSC) emulsified in complete Freund's adjuvant were used for both induction and reinduction of EAE. The results showed that: a) sex has no influence on clinical signs of EAE in young DA rats aged 5 or 8 weeks; b) susceptibility to EAE induction increases with age; c) RSC is a more potent encephalitogen than GPSC; d) GPSC is a more effective antigen for anti-MBP antibody production than RSC; e) anti-MBP antibody levels are not correlated with clinical score of EAE; f) EAE can be reinduced in 27-week- old rats if RSC is used for induction and/or reinduction; and g) anti-MBP antibodies are not related to resistance to EAE reinduction in DA rats.U radu su prikazani rezultati ispitivanja osetljivosti na indukciju i reindukciju eksperimentalnog alergijskog encefalomojelitisa (EAE) u DA pacova, oba pola, starosti 5,8 i 27 nedelja. Za imunizaciju su korišćeni homogenati kičmene moždine zamorca (KMZ) ili kičmene moždine Lewis pacova (KMP) u kompletnom Freund-ovom adjuvansu. Rezultati su pokazali da: a) pol ne utiče na razvoj EAE-a u 5 i 8 nedelja starih DA pacova; b) osetljivost na EAE raste sa starošću; c) KMP je jači encefalitogen od KMZ; d) produkcija anti-mijelin bazni protein (MBP) antitela je veća nakon imunizacije sa KMZ nego sa KMP; e) nivo anti-MBP antitela nije u korelaciji sa kliničkim znakom EAE-a; f) reindukcija EAE-a je moguća u pacova starih 27 nedelja ako se za indukciju i/ili reindukciju koristi KMP; i g) anti-MBP antitela nisu odgovorna za rezistenciju na reindukciju EAE-a u DA pacova

Karakteristike antigena utiču na efekte stresa na humoralni imunski odgovor u pacova

The objective of the present study was to explore whether the suppressive effect of electric stress (ES) on the immune response in rats was limited to the particular antigen given concomitantly with ES. Therefore, the influence of simultaneous exposure to stress and immunization with an unrelated antigen (keyhole limpet hemocyanin, KLH) on the humoral immune response to bovine serum albumin (BSA) was investigated. Specific anti-KLH antibody levels were also determined in rats exposed to ES and concomitantly immunized with BSA. Five daily sessions of ES or immunization with KLH 2 weeks prior to immunization with BSA did not influence the secondary humoral immune response to BSA, but concomitant exposure to ES and immunization with KLH significantly decreased it. Conversely, the primary humoral immune response to KLH was suppressed by exposure of the animals to ES at the time of immunization with KLH, as well as at the time of the immunization with BSA 2 weeks later. It is suggested that the suppressive effect of ES on the humoral immune response is not specific for a certain antigen. However, the chemical and immunological characteristics of the antigens shaped the profile of stress-induced immune changes with respect to the sensitivity of the primary and secondary immune response and the duration of the effect.Cilj rada bio je da se utvrdi da li je supresivni efekat električnog stresa (ES) na imunski odgovor specifičan za antigen kojim su pacovi imunizovani u vreme izlaganja ES. Ispitivan je uticaj istovremene primene stresa i imunizacije sa nesrodnim antigenom (keyhole limpet hemocyanin, KLH) na humoralni imunski odgovor prema goveđem serum albuminu (GSA). Takođe su određivana i specifična anti-KLH antitela u serumima pacova koji su bili istovremeno izloženi stresu i imunizovani sa GSA. Rezultati su pokazali da ni petodnevni ES, ni imunizacija sa KLH dve nedelje pre imunizacije sa GSA nisu uticali na nivo anti- GSA antitela, za razliku od istovremenog izlaganja ES i imunizacije sa GSA koji su značajno suprimirali sekundarni humoralni imunski odgovor prema GSA. Nasuprot tome, primarni humoralni imunski odgovor prema KLH je bio suprimiran u pacova koji su bili izloženi stresu tokom imunizacije sa KLH, ali i kod onih koji su bili izloženi stresu dve nedelje kasnije odnosno tokom imunizacije sa GSA. Naši rezultati ukazuju da supresivni efekat stresa na imunski odgovor nije specifičan za određen antigen, kao i da hemijske i imunološke osobine antigena značajno utiču na kvalitet promena izazvanih stresom u pogledu osetljivosti primarnog i sekundarnog imunskog odgovora na stres i trajanja efekata stresa

Produkcija H2O2 i NO peritonealnih makrofaga pacova u odgovoru na crevne komensalne bakterije

The importance of commensal bacteria in the immune system development and its involvement in the etiopatogenetic mechanisms of complex multifactorial and multigenic diseases is well documented. The aim of the present study was to compare the levels of hydrogen peroxide (H2O2) and nitric oxide (NO) produced by resident peritoneal macrophages from the autoimmune disease susceptible Dark Agouti (DA) rats vs. resistant Albino Oxford (AO) rat strain, under basal conditions and subsequent to in vitro stimulation with gut commensals. Following the stimulation with phorbol myristil acetate (PMA), E. coli/PMA or P. mirabilis/PMA, AO rats macrophages have produced significantly higher levels of H2O2 compared to the cells from DA rats. Strain differences in NO production were not detected under basal conditions and after the stimulation with lipopolysaccharide and P. mirabilis. However, after the in vitro stimulation with E. coli, AO rats macrophages have produced higher levels of NO compared to DA rats macrophages. Our results demonstrated that macrophages from AO rats have higher potential to produce H2O2 and NO in response to specific commensal bacteria when compared to DA rats. A possible relationship between the macrophage activity in response to commensal bacteria and the susceptibility to induction of autoimmune/inflammatory diseases in AO and DA rat strains is suggested.Poznato je da komensalna crevna flora ima značajnu ulogu u razvoju imunskog sistema kao i u etiopatogenezi kompleksnih multifaktorijalnih i multigenetskih bolesti. Cilj ovog rada bio je da se uporedi produkcija vodonik peroksida (H2O2) i azot monoksida (NO) peritonealnih makrofaga dva inbredna soja pacova, od kojih je jedan osetljiv (Dark Agouti, DA), a drugi rezistentan (Albino Oxford, AO) na indukciju autoimunskih bolesti, kako u bazalnim uslovima tako i nakon in vitro stimulacije makrofaga sa crevnim komensalima. Nakon stimulacije sa forbol miristil acetatom (PMA), E. coli/PMA and P. mirabilis/PMA makrofage AO pacova su produkovale značajno više H2O2 u poređenju sa makrofagama DA pacova. Nisu detektovane sojne razlike u produkciji NO u bazalnim uslovima, kao ni posle stimulacije sa lipopolisaharidom i P. mirabilis. Međutim, nakon in vitro stimulacije sa E. coli makrofage AO pacova su produkovale više NO u odnosu na makrofage DA pacova. Naši rezultati su ukazali da makrofage AO pacova imaju veći potencijal za produkciju H2O2 i NO u odgovoru na specifične komensalne bakterije. Ova različita aktivnost makrofaga može biti u vezi sa različitom osetljivošću na indukciju autoimunskih/inflamatornih bolesti kod DA i AO soja pacova

Lactobacillus rhamnosus Affects Rat Peritoneal Cavity Cell Response to Stimulation with Gut Microbiota: Focus on the Host Innate Immunity

Gut microbiota contribute to shaping the immune repertoire of the host, whereas probiotics may exert beneficial effects by modulating immune responses. Having in mind the differences in both the composition of gut microbiota and the immune response between rats of Albino Oxford (AO) and Dark Agouti (DA) rat strains, we investigated if intraperitoneal (i.p.) injection of live Lactobacillus rhamnosus (LB) may influence peri-toneal cavity cell response to invitro treatments with selected microbiota in the rat strain-dependent manner. Peritoneal cavity cells from AO and DA rats were lavaged two (d2) and seven days (d7) following i.p. injection with LB and tested for NO, urea, and H2O2 release basally, or upon invitro stimulation with autologous E.coli and Enterococcus spp. Whereas the single i.p. injection of LB nearly depleted resident macrophages and increased the proportion of small inflammatory macrophages and monocytes on d2 in both rat strains, greater proportion of MHCIIhiCD163− and CCR7+ cells and increased NO/diminished H2O2 release in DA compared with AO rats suggest a more intense inflammatory prim-ing by LB in this rat strain. Even though E.coli- and/or Enterococcus spp.-induced rise in H2O2 release invitro was abrogated by LB in cells from both rat strains, LB prevented microbiota-induced increase in NO/urea ratio only in cells from AO and augmented it in cells from DA rats. Thus, the immunomodulatory properties may not be constant for particular probiotic bacteria, but shaped by innate immunity of the host.https://rdcu.be/cxyn

Strain differences and the role for HSP47 and HSP70 in adjuvant arthritis in rats

Because of high sequence homology between microbial and endogenous heat shock proteins (HSP), immunological cross-reactivity to microbial HSP has been suggested as a possible cause of the development of autoimmune diseases, such as rheumatoid arthritis. The present study aimed to determine a potential role of HSP47, a molecular chaperone involved in the synthesis and assembly of collagen molecules, and microbial HSP71 (mHSP71) in adjuvant arthritis (AA) in two rat strains: Dark Agouti (DA), susceptible to AA induction and Albino Oxford (AO), which is resistant to AA induction. Immunization with complete Freund's adjuvant (CFA) induced an increased expression of HSP47 in joints of DA rats, which exhibited severe clinical signs of AA at the time of disease peak, while this protein was not detectable in joints of AO rats. In contrast, no strain differences in HSP72 (rat analogue of mHSP71) expressions in joints were observed. The increased levels of anti-HSP47 antibodies were detected in sera of DA rats during the AA peak, while the immunization with CFA increased levels of anti-mHSP71 antibodies in sera of AO rats. HSP47 and mHSP71 reduced proliferation of draining inguinal lymph node cells (LNC) in resistant AO rat strain, leading to a hypothesis that both HSP participated in AA control. Finally, mHSP71 potentiated the apoptotic response of LNC in susceptible DA rat strain. In conclusion, our findings indicate involvement of HSP47 in the development of AA in the rat, and point out to the regulatory role for both HSP47 and mHSP71

Fenotipske promene izazvane imunizacijom encefalitogenom menjaju funkcije peritonealnih makrofaga u dva soja pacova različite osetljivosti prema indukciji eksperimentalnog autoimunskog encefalomijelitisa (EAE).

We have investigated the phenotype of peritoneal cells and the functions of peritoneal macrophages obtained from experimental autoimmune encephalomyelitis (EAE)-susceptible Dark Agouti (DA) and EAE-resistant Albino Oxford (AO) rat strains on days 1, 3 and 7 post immunization with encephalitogen. Resident peritoneal cells from immunized and non-immunized rats of both strains were subjected to flow cytometric analyzes and after adherence were tested for zymosan phagocytosis, hydrogen peroxide (H2O2) and nitric oxide (NO) production. In non-immunized rats, macrophages from the DA rat strain phagocytosed more zymosan but produced less H2O2 than cells from the AO strain, while both strains produced comparable amounts of NO. Immunization increased phagocytosis in DA rats' cells, but decreased both phagocytosis and H2O2 production in cells from AO rats. Overall higher phagocyte ability in DA rats was associated with a significantly larger population of ED1+ cells (macrophages and dendritic cells), in contrast to a more pronounced expression of ED2 antigen (resident macrophages) on cells from AO rats. Immunization also increased the expression of CD11b molecule on non-resident ED2-macrophages of DA, but not of AO rats. The early and subtle phenotype changes in peritoneal cells of both rat strains might mirror the mechanism contributing to their different sensitivity to the induction of autoimmunity.Ispitivan je fenotip peritonealnih ćelija, kao i funkcije peritonealnih makrofaga, izolovanih od pacova Dark Agouti (DA) soja osetljivog na indukciju eksperimentalnog autoimunskog encefalomijelitisa (EAE) i pacova Albino Oxford (AO) soja koji je rezistentan prema EAE-u, 1, 3. i 7. dana nakon imunizacije encefalitogenom. Rezidentne peritonealne ćelije su ispitivane metodom protočne citofluorometrije, a zatim je nakon adherence testirana njihova sposobnost fagocitoze čestica zimozana i kapacitet produkcije vodonik peroksida (H2O2) i azot monoksida (NO). U neimunizovanih pacova makrofage DA soja su intenzivnije fagocitovale čestice zimozana i imale nižu sposobnost produkcije H2O2 nego ćelije pacova AO soja, ali nije bilo sojnih razlika u sposobnosti produkcije NO. Imunizacija je dovela do povećanja fagocitne sposobnosti makrofaga DA pacova, ali i do smanjenja fagocitoze i produkcije H2O2 makrofaga pacova AO soja. Generalno veću sposobnost fagocitoze u DA pacova prati i značajno veća zastupljenost ED1+ ćelija (koje čine uglavnom makrofage i dendritične ćelije) nasuprot većoj zastupljenosti ED2 antigena (marker rezidentnih makrofaga) na ćelijama pacova AO soja. Imunizacija encefalitogenom je takođe dovela do povećanja ekspresije CD11b molekula na nerezidentnim ED2- ćelijama pacova DA, ali ne i AO soja. Rane i diskretne fenotipske promene na peritonealnim ćelijama pacova oba soja verovatno odslikavaju mehanizme koji doprinose njhovoj različitoj osetljivosti prema indukciji autoimunskih oboljenja

Razlike u edemu šape pacova indukovanom konkanavalinom a u zavisnosti od soja - uticaj histaminskih H1 i H2 receptora

The present study tests the hypothesis that the difference in the intensity of paw edema found between the Dark Agouti (DA) and Albino Oxford (AO) rat strains originates from the distinct participation of histamine, serotonin and their corresponding receptors in Concanavalin A (Con A)-induced inflammation. DA and AO male rats were intraplantarly injected with specific receptor antagonists prior to Con A, and the intensity of inflammation was determined by measuring the paw diameter. Our results have showed that histamine H1 and H2 receptor antagonists reduced the Con A-induced paw edema in DA rats, while serotonin 5HT3 receptor antagonist diminished the inflammation in both DA and AO rat strains. The calcium channel blocker did not change Con A-induced inflammation. Strain differences in the intensity and kinetics of inflammation observed between the DA and AO rats are most likely defined by the diversity of mediators released and their receptors activated upon Con A injection.Testirana je hipoteza da razlike u intenzitetu inflamatornog edema šape indukovanog konkanavalinom A u pacova Dark Agouti (DA) i Albino Oxford (AO) soja potiču od različitog doprinosa histamina i serotonina i njihovih odgovarajućih receptora. Mužjaci pacova DA i AO soja su intraplantarno tretirani antagonistima specifičnih receptora pre izazivanja inflamacije konkanavalinom A i intenzitet inflamacije je praćen merenjem dijametra šape. Naši rezultati su ukazali da antagonisti histaminskih H1 i H2 receptora smanjuju edem šape indukovan konkanavalinom A u DA pacova, dok antagonist serotoninskih 5HT3 receptora smanjuje edem šape u oba soja pacova. Blokator kalcijumskih kanala ne utiče na inflamaciju izazvanu konkanavalinom A. Razlike u intenzitetu i kinetici inflamatornog odgovora indukovanog konkanavalinom A između DA i AO sojeva su najverovatnije posledica razlika u oslobođ enim medijatorima i aktivaciji odgovarajućih receptora nakon injekcije konkanavalina A

Peritoneal exudate cells from long-lived rats exhibit increased IL-10/IL-1 beta expression ratio and preserved NO/urea ratio following LPS-stimulation in vitro

In humans, usual aging, differently from successful aging, is associated with deregulation of proinflammatory/anti-inflammatory cytokine balance. The corresponding data from rat studies are limited. Therefore, we examined (i) cytokine messenger RNA (mRNA) profile of fresh peritoneal cells from 6-(adult), 24-(old), and 31-month-old (long-lived) AO rats and (ii) proinflammatory (IL-1 beta and IL-6) and antiinflammatory (IL-10) cytokine, NO, and urea production in their LPS-stimulated cultures. Comparing with adult rats, cells from old ones expressed lower amount of TNF-alpha and IL-6 mRNAs, but greater amount of IL-1 beta mRNA. On the other hand, cells fromlong-lived rats exhibited a dramatic increase in IL-10 mRNA expression followed by diminished TNF-alpha and IL-6 mRNA expression, and comparable expression of IL-1 beta mRNA relative to adult rats. Consequently, IL-10/IL-1 beta mRNA ratio was greater in cells from long-lived rats than in adult and old rats. In LPS-stimulated peritoneal cell cultures (contained = 95 % macrophages) from old rats, concentration of common proinflammatory cytokines was higher than in those from adult rats. Comparing with adult and old rats, in LPS-stimulated macrophage cultures from long-lived rats, TNF-alpha and IL-6 concentrations were lower; IL-1 beta concentration was comparable or greater (in respect to adult rats), whereas that of IL-10 was strikingly higher. Consistently, in macrophage cultures from long-lived rats, NO (iNOS activity marker)/urea (arginase activity marker) ratio was less and not different from that in old and adult rats, respectively. The study suggests that macrophages from longlived rats, differently from those of old ones, have substantial ability to limit proinflammatory mediator production, which may contribute to their longevity

17β-Estradiol and genistein affect macrophage inflammatory cytokine production during aging in sex-specific manner

Aging differently affects the expression of estrogen receptors alpha and beta (ERα and ERβ) and Toll-like receptors (TLR4) on peritoneal cavity cells of male and female rats. We explored the involvement of ERα and ERβ in the in vitro treatment of LPS-stimulated peritoneal macrophages with 17β-estradiol (which stimulates both receptors) or genistein (which is predominantly an ERβ agonist) on inflammatory cytokine secretion from young (3 months old) and middle-aged (16 months old) female and male AO rats. Aging diminished the proportion of TLR4+ cells and secretion of IL-1β and IL-6 in macrophages from female rats while the effect on male rat macrophages was opposite. 17βestradiol increased IL-1β secretion by middle-aged females’ macrophages via ERα, and suppressed it in cells from young females via ERβ. Genistein-induced decrease of IL-1β in macrophages from all experimental groups was probably mediated by ERβ. 17β-estradiol augmented IL-6 secretion by cells from all experimental groups via ERα while genistein diminished it in all females’ and in middle-aged male rats’ macrophages by activating ERβ. However, genistein increased IL-6 secretion from macrophages of young male rats via ERα. Although 17β-estradiol and genistein stimulated secretion of macrophage inflammatory cytokines via ERα and suppressed it probably via ERβ, their modulatory actions were determined by aging-induced changes in macrophage ERs expression and possible ERα / ERβ interactions (Supported by Ministry of Education, Science and Technological development, Republic of Serbia, Grant No 175050)

Potential impact of early-life probiotic supplementation on peritoneal macrophage function

Clinical and animal trials show that early life probiotic consumption provides health benefits in adult life by modulating the immune response. We tested the effects of early life oral consumption of the probiotic Lactobacillus rhamnosus on the function and phenotype of rat peritoneal cavity cells in a model of induced colitis. For the first month of their lives, rats were either fed with an aqueous probiotic bacteria suspension (LB group) or tap water (control group). When the rats grew to 3 months old, we studied the response of their peritoneal macrophages to autologous fecal bacteria stimulation in vitro, both before and after colitis induction (TNBS 40mg/kg of body mass in 50% ethanol). Compared to the controls, the peritoneal cavity cells of the LB group produced less nitric oxide (NO) and had an increased proportion of CD163+ cells. The rats in the LB group have shown milder symptoms of colitis (shorter length of colon under necrosis, less severe submucosal infiltration, lesser degree of colonic wall thickening), along with a diminished increase of peritoneal proinflammatory CCR7+ cells and blunted NO production in response to stimulation by autologous fecal bacteria. Our results may indicate that early oral probiotic administration attenuates macrophage responses to fecal bacteria, which are the primary cause of tissue inflammation and necrosis in chemically induced colitis models, and that this attenuation may be involved in improving the health of colitic rats