Lack of Th17 cell generation in patients with severe burn injuries

Immunodeficient patients with severe burn injuries are extremely susceptible to infection with Candida albicans. In addition to Th1 cells, IL-17-producing CD4(+) T cells (Th17 cells) have recently been described as an important effector cell in host anti-Candida resistance. In this study, therefore, we tried to induce Th17 cells in cultures of severely burned patient PBMC by stimulation with the C. albicans Ag (CAg). In the results, the biomarkers for Th17 cells (IL-17 production and intracellular expression of IL-17 and retinoic acid receptor-related orphan receptor γt) were not displayed by burn patient PBMC stimulated with CAg, whereas these biomarkers of Th17 cells were detected in cultures of healthy donor PBMC stimulated with CAg. Burn patient sera were shown to be inhibitory on CAg-stimulated Th17 cell generation in healthy donor PBMC cultures; however, Th17 cells were induced by CAg in healthy donor PBMC cultures supplemented with burn patient sera that were previously treated with anti-IL-10 mAb. Also, the biomarkers of Th17 cells were not induced by CAg in healthy donor PBMC cultures supplemented with rIL-10. IL-10 was detected in serum specimens derived from severely burned patients. These results indicate that Th17 cells are not generated in burn patient PBMC cultures supplemented with CAg. IL-10, produced in response to burn injuries, is shown to be inhibitory on Th17 cell generation. The high susceptibility of severely burned patients to C. albicans infection might be influenced if burn-associated IL-10 production is intervened.     

Peripheral Monocyte Functions and Activation in Patients with Quiescent Crohn’s Disease

Recent developments suggest a causal link between inflammation and impaired bacterial clearance in Crohn’s disease (CD) due to alterations of intestinal macrophages. Studies suggest that excessive inflammation is the consequence of an underlying immunodeficiency rather than the primary cause of CD pathogenesis. We characterized phenotypic and functional features of peripheral blood monocytes of patients with quiescent CD (n = 18) and healthy controls (n = 19) by analyses of cell surface molecule expression, cell adherence, migration, chemotaxis, phagocytosis, oxidative burst, and cytokine expression and secretion with or without lipopolysaccharide (LPS) priming. Peripheral blood monocytes of patients with inactive CD showed normal expression of cell surface molecules (CD14, CD16, CD116), adherence to plastic surfaces, spontaneous migration, chemotaxis towards LTB4, phagocytosis of E. coli, and production of reactive oxygen species. Interestingly, peripheral blood monocytes of CD patients secreted higher levels of IL1β (p<.05). Upon LPS priming we found a decreased release of IL10 (p<.05) and higher levels of CCL2 (p<.001) and CCL5 (p<.05). The expression and release of TNFα, IFNγ, IL4, IL6, IL8, IL13, IL17, CXCL9, and CXCL10 were not altered compared to healthy controls. Based on our phenotypic and functional studies, peripheral blood monocytes from CD patients in clinical remission were not impaired compared to healthy controls. Our results highlight that defective innate immune mechanisms in CD seems to play a role in the (inflamed) intestinal mucosa rather than in peripheral blood.     

Effect of Glycyrrhizin on Pseudomonal Skin Infections in Human-Mouse Chimeras

In our previous studies, peripheral blood lineage⁻CD34⁺CD31⁺ cells (CD31⁺ IMC) appearing in severely burned patients have been characterized as inhibitor cells for the production of β-defensins (HBDs) by human epidermal keratinocytes (NHEK). In this study, the effect of glycyrrhizin on pseudomonal skin infections was studied in a chimera model of thermal injury. Two different chimera models were utilized. Patient chimeras were created in murine antimicrobial peptide-depleted NOD-SCID IL-2rγ^null mice that were grafted with unburned skin tissues of severely burned patients and inoculated with the same patient peripheral blood CD31⁺ IMC. Patient chimera substitutes were created in the same mice that were grafted with NHEK and inoculated with experimentally induced CD31⁺ IMC. In the results, both groups of chimeras treated with glycyrrhizin resisted a 20 LD₅₀ dose of P. aeruginosa skin infection, while all chimeras in both groups treated with saline died within 3 days of the infection. Human antimicrobial peptides were detected from the grafted site tissues of both groups of chimeras treated with glycyrrhizin, while the peptides were not detected in the same area tissues of controls. HBD-1 was produced by keratinocytes in transwell-cultures performed with CD31⁺ IMC and glycyrrhizin. Also, inhibitors (IL-10 and CCL2) of HBD-1 production by keratinocytes were not detected in cultures of patient CD31⁺ IMC treated with glycyrrhizin. These results indicate that sepsis stemming from pseudomonal grafted site infections in a chimera model of burn injury is controllable by glycyrrhizin. Impaired antimicrobial peptide production at the infection site of severely burned patients may be restored after treatment with glycyrrhizin.     

Interleukin-15 modulates interferon-gamma and beta-chemokine production in patients with HIV infection: implications for immune-based therapy

Interleukin (IL)-15 is a cytokine that has lymphocyte stimulatory activity similar to that of IL-2, and plays important immunoregulatory functions during HIV disease. To evaluate the role of IL-15 in HIV infection the following patients were studied: 18 antiretroviral-naive patients with advanced disease; 19 patients with continuous viral suppression and immunological response after 48-120 weeks of highly active antiretroviral therapy (HAART); and 12 patients with evidence of virological and immunological HAART treatment failure. Nineteen healthy blood donors were included as controls. The production of IL-15 by human peripheral blood monocytes stimulated with lipopolysaccharide and Mycobacterium avium complex, the priming effect of IL-15 on IFN-gamma production from purified CD4(+) and CD8(+) T cells, and the ability of IL-15 to stimulate the beta-chemokine release from purified CD4(+) and CD8(+) T cells were analyzed. In the present work IL-15 production by human peripheral blood monocytes was significantly increased in HIV-infected patients with long-term virological and immunological response to HAART. IL-15 enhanced the in vitro priming of CD4(+) and CD8(+) T cells for IFN-gamma production, also in patients receiving HAART. Finally, IL-15 had positive effects on RANTES, MIP-1alpha, and MIP-1beta release by CD4(+) and CD8(+) T cells. In conclusion IL-15 could affect the immune response of HIV-infected patients by augmenting and/or modulating IFN-gamma production and beta-chemokine release. These data about functional properties of IL-15 could provide new implications for immune-based therapies in HIV infection.     

Impaired ability of burn patient neutrophils to stimulate β-defensin production by keratinocytes

Immunosuppressive neutrophils (PMN-II) appearing in association with burn injury have a role on the increased susceptibility of burn patients to various infections. In the present study, the role of PMN-II on the production of human β-defensins (HBDs), important molecules on host antimicrobial innate immunities, by human keratinocytes was studied. Normal human epidermal keratinocytes (NHEKs) were cultured with neutrophils (PMNs) isolated from burn patients or healthy volunteers in dual-chamber transwells. Culture fluids harvested 24?h after cultivation were assayed for HBDs using enzyme-linked immunosorbent assay. Also, these culture fluids were assayed for their antimicrobial activities by a standard colony-counting method using Pseudomonas aeruginosa. In the results, PMNs isolated from peripheral blood of burn patients were confirmed as PMN-II, because these cells produced CC-chemokine ligand 2 (CCL2), but not interleukin (IL)-12 and CC-chemokine ligand 3 (CCL3). Culture fluids of NHEK transwell-cultured with healthy PMNs exhibited strong killing activities against P. aeruginosa (96% inhibition), however, the growth of bacteria was not dramatically inhibited by the culture fluids of NHEK transwell-cultured with burn-patient PMNs (36% inhibition). IL-12 and CCL3 containing culture fluids of healthy PMNs stimulated with the bacterial antigen or rCCL3 and rIL-12 enhanced the production of HBD2 and HBD3 by NHEK, whereas CCL2 containing culture fluids of burn-patient PMN stimulated with the antigen or rCCL2 inhibited the HBD production by NHEK. These results indicate that PMN-II appearing in association with burn injury contribute to the decreased production of HBDs in thermally injured patients.     

Colonic eosinophilic inflammation in experimental colitis is mediated by Ly6C(high) CCR2(+) inflammatory monocyte/macrophage-derived CCL11

Recent genome-wide association studies of pediatric inflammatory bowel disease have implicated the 17q12 loci, which contains the eosinophil-specific chemokine gene CCL11, with early-onset inflammatory bowel disease susceptibility. In the current study, we employed a murine model of experimental colitis to define the molecular pathways that regulate CCL11 expression in the chronic intestinal inflammation and pathophysiology of experimental colitis. Bone marrow chimera experiments showed that hematopoietic cell-derived CCL11 is sufficient for CCL11-mediated colonic eosinophilic inflammation. We show that dextran sodium sulfate (DSS) treatment promotes the recruitment of F4/80(+)CD11b(+)CCR2(+)Ly6C(high) inflammatory monocytes into the colon. F4/80(+)CD11b(+)CCR2(+)Ly6C(high) monocytes express CCL11, and their recruitment positively correlated with colonic eosinophilic inflammation. Phenotypic analysis of purified Ly6C(high) intestinal inflammatory macrophages revealed that these cells express both M1- and M2-associated genes, including Il6, Ccl4, Cxcl2, Arg1, Chi3l3, Ccl11, and Il10, respectively. Attenuation of DSS-induced F4/80(+)CD11b(+)CCR2(+)Ly6C(high) monocyte recruitment to the colon in CCR2(-/-) mice was associated with decreased colonic CCL11 expression, eosinophilic inflammation, and DSS-induced histopathology. These studies identify a mechanism for DSS-induced colonic eosinophilia mediated by Ly6C(high)CCR2(+) inflammatory monocyte/macrophage-derived CCL11.     

Increase of expression and activation of chemokine CCL15 in chronic renal failure

Chemokines are believed to be involved in the pathogenesis of chronic renal failure (CRF). In CRF, significantly increased CCL15-IR plasma concentrations were detected. Whereas in plasma of healthy individuals one predominant CCL15-IR molecule with a M(w) of 15kDa [high molecular weight (HMW-CCL15-IR)] was identified, CRF plasma contains increased concentrations of truncated CCL15-IR molecules [intermediate molecular weight (IMW-CCL15-IR)]. HMW-CCL15-IR isolated from hemofiltrate revealed an M(w) of 10141.3, corresponding to deglycosylated CCL15(1-92) carrying a N-terminal pyrrolidone carboxylic acid. CCL15(12-92) was identified as a major component of IMW-CCL15-IR in CRF plasma. Compared to CCL15(1-92), in monocytes CCL15(12-92) causes stronger induction of intracellular calcium flux, chemotactic activity, and adhesion to fibronectin. Intracellular calcium flux assays revealed that, in comparison to peripheral blood mononuclear cells (PBMC) of healthy donors, PBMCs of CRF patients demonstrated an increased sensitivity to CCL15. Our results point to an involvement of the CCL15-CCR1 axis in the pathophysiology of CRF.     

Effect of azacytidine in the release of leukemia inhibitory factor,oncostatin m,interleukin (IL)-6, and IL-11 by mononuclear cells of patients with refractory anemia

5-azacytidine (AZA) yields hematologic improvement in patients with myelodysplastic syndromes (MDS). Ineffective hemopoiesis in MDS produce the paradox of high intramedullary cellularity with peripheral cytopenias. Leukemia inhibitory factor (LIF), oncostatin M (OSM), interleukin (IL)-6, and IL-11 regulate hemopoiesis and LIF, OSM, and IL-6 also inhibit the proliferation of myeloid leukemic cell lines through the signal-transducing subunit gp130. These IL-6-type cytokines were measured by enzyme-linked immunosorbent assay in cell culture supernatants (SN) obtained from peripheral blood mononuclear cells (MNC) and monocyte-depleted MNC of patients with refractory anemia (RA; n=12) and healthy individuals (n=10). AZA down-regulated OSM, IL-6, and IL-11 release by MNC of patients but not by MNC from healthy individuals. Patient's SN had significantly lower concentrations of LIF, OSM, and IL-11 than SN of normal subjects. When monocyte-depleted MNC of patients were stimulated with phytohemagglutinin a significant increment in OSM levels was observed. In contrast, monocyte depletion in healthy subjects did not cause any significant change in OSM values. We conclude that: (a) AZA inhibits the release of OSM, IL-6, and IL-11 exclusively in RA-diseased MNC, (b) Patient's MNC release subnormal amounts of LIF, OSM, and IL-11, and (c) RA-derived monocytes probably down-regulate OSM release by phytohemagglutinin-activated MNC.     

Functional characterization of cultured keratinocytes after acute cutaneous burn injury

Background

In addition to forming the epithelial barrier against the outside environment keratinocytes are immunologically active cells. In the treatment of severely burned skin, cryoconserved keratinocyte allografts gain in importance. It has been proposed that these allografts accelerate wound healing also due to the expression of a favourable - keratinocyte-derived - cytokine and growth factor milieu.

Methods

In this study the morphology and cytokine expression profile of keratinocytes from skin after acute burn injury was compared to non-burned skin. Skin samples were obtained from patients after severe burn injury and healthy controls. Cells were cultured and secretion of selected inflammatory mediators was quantified using Bioplex Immunoassays. Immunohistochemistry was performed to analyse further functional and morphologic parameters.

Results

Histology revealed increased terminal differentiation of keratinocytes (CK10, CK11) in allografts from non-burned skin compared to a higher portion of proliferative cells (CK5, vimentin) in acute burn injury. Increased levels of IL-1α, IL-2, IL-4, IL-10, IFN-γ and TNFα could be detected in culture media of burn injury skin cultures. Both culture groups contained large amounts of IL-1RA. IL-6 and GM-CSF were increased during the first 15 days of culture of burned skin compared to control skin. Levels of VEGF, FGF-basic, TGF-ß und G-CSF were high in both but not significantly different. Cryoconservation led to a diminished mediator synthesis except for higher levels of intracellular IL-1α and IL-1ß.

Conclusion

Skin allografts from non-burned skin show a different secretion pattern of keratinocyte-derived cytokines and inflammatory mediators compared to keratinocytes after burn injury. As these secreted molecules exert auto- and paracrine effects and subsequently contribute to healing and barrier restoration after acute burn injury therapies affecting this specific cytokine/growth factor micromilieu could be beneficial in burned patients.     

Restoration of natural IgM production from liver B cells by exogenous IL-18 improves the survival of burn-injured mice infected with Pseudomonas aeruginosa

Pseudomonas aeruginosa is the most common bacterium of postburn infection. In the present study we investigated the immune mechanism of susceptibility to this type of postburn infection and also examined the efficacy of IL-18 treatment. C57BL/6 mice were challenged with P. aeruginosa on day 7 after burn injury. Although the burn-injured mice showed a poor survival rate after bacterial challenge, they retained their IFN-gamma production. The burned mice showed lower serum IgM levels and a poor IgM response following P. aeruginosa challenge in comparison with the sham mice, whereas IL-18 treatment after burn injury (alternate day injections for 1 wk) greatly improved the serum IgM levels, which are P. aeruginosa-independent natural IgM before bacterial challenge, thereby increasing the survival rate after the challenge. IL-18 treatment also induced specific IgM to P. aeruginosa in the sera 5 days after bacterial challenge in the burned mice. Interestingly, CD43(+)CD5(-)CD23(-)B220(dim) cells, namely B-1b cells, increased in the liver after the IL-18 treatment and were found to actively produce IgM in vitro without any additional stimulation. Furthermore, the IL-18 treatment up-regulated the neutrophil count and the C3a levels in the blood as a result of the increased IgM level, which may thus play a critical role in the opsonization and elimination of any invading bacteria. IL-18 treatment for the burned mice and their resultant natural IgM production were thus found to strengthen the host defense against P. aeruginosa infection.     

Interleukin-19 Impairment in Active Crohn’s Disease Patients

The exact function of interleukin-19 (IL-19) on immune response is poorly understood. In mice, IL-19 up-regulates TNFα and IL-6 expression and its deficiency increases susceptibility to DSS-induced colitis. In humans, IL-19 favors a Th2 response and is elevated in several diseases. We here investigate the expression and effects of IL-19 on cells from active Crohn’s disease (CD) patient. Twenty-three active CD patients and 20 healthy controls (HC) were included. mRNA and protein IL-19 levels were analyzed in monocytes. IL-19 effects were determined in vitro on the T cell phenotype and in the production of cytokines by immune cells. We observed that unstimulated and TLR-activated monocytes expressed significantly lower IL-19 mRNA in active CD patients than in HC (logFC = −1.97 unstimulated; −1.88 with Pam3CSK4; and −1.91 with FSL-1; p<0.001). These results were confirmed at protein level. Exogenous IL-19 had an anti-inflammatory effect on HC but not on CD patients. IL-19 decreased TNFα production in PBMC (850.7±75.29 pg/ml vs 2626.0±350 pg/ml; p<0.01) and increased CTLA4 expression (22.04±1.55% vs 13.98±2.05%; p<0.05) and IL-4 production (32.5±8.9 pg/ml vs 13.5±2.9 pg/ml; p<0.05) in T cells from HC. IL-10 regulated IL-19 production in both active CD patients and HC. We observed that three of the miRNAs that can modulate IL-19 mRNA expression, were up-regulated in monocytes from active CD patients. These results suggested that IL-19 had an anti-inflammatory role in this study. Defects in IL-19 expression and the lack of response to this cytokine could contribute to inflammatory mechanisms in active CD patients.     

CCL18 is expressed in atopic dermatitis and mediates skin homing of human memory T cells

CCL18 is a human chemokine secreted by monocytes and dendritic cells. The receptor for CCL18 is not yet known and the functions of this chemokine on immune cells are not fully elucidated. In this study, we describe that CCL18 is present in skin biopsies of atopic dermatitis (AD) patients but not in normal or psoriatic skin. CCL18 was specifically expressed by APCs in the dermis and by Langerhans and inflammatory dendritic epidermal cells in the epidermis. In addition, the serum levels of CCL18 and the percentages of CCL18-producing monocyte/macrophages and dendritic cells were significantly increased in AD patients compared with healthy controls. Furthermore, we demonstrate that CCL18 binds to CLA(+) T cells in peripheral blood of AD patients and healthy individuals and induces migration of AD-derived memory T cells in vitro and in human skin-transplanted SCID mice. These findings highlight a unique role of CCL18 in AD and reveal a novel function of this chemokine mediating skin homing of a subpopulation of human memory T cells.     

Red Blood Cell-Conditioned Media from Non-Alcoholic Fatty Liver Disease Patients Contain Increased MCP1 and Induce TNF-α Release

Background:Non-alcoholic fatty liver disease (NAFLD) constitutes a global pandemic. An intricate network among cytokines and lipids possesses a central role in NAFLD pathogenesis. Red blood cells comprise an important source of both cytokines and signaling lipids and have an important role in molecular crosstalk during immunometabolic deregulation. However, their role in NAFLD has not been thoroughly investigated.Methods:Conditioned media from erythrocytes derived from 10 NAFLD patients (4 men, 6 women, aged 57.875±15.16) and 10 healthy controls (4 men, 6 women, aged 39.3±15.55) was analyzed for the cytokines IFN-γ, TNF-α, CCL2, CCL5, IL-8, IL-1β, IL-12p40, IL-17, MIP-1β, the signaling lipids sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA), and cholesterol. Their effect on the cytokine profile released by RAW 264.7 macrophages was also studied.Results:MCP1 levels were greater in conditioned growth medium from NAFLD patient erythrocytes than in that from healthy controls (37±40 vs 6.51±5.63 pg/ml). No statistically significant differences were found between patients and healthy controls with regard to S1P, LPA, cholesterol, or eight other cytokines. TNF-a release by RAW 264.7 cells was greater after incubation with patient-derived erythrocyte-conditioned medium than in medium without RAW 264.7 cells from either healthy or NAFLD subjects.Conclusion:Erythrocytes may contribute to liver infiltration by monocytes, and macrophage activation, partially due to CCL2 release, in the context of NAFLD..Key Words: Cytokines, Erythrocytes, Lipids, Non-alcoholic fatty liver disease, Signaling     

Glycyrrhizin inhibits the manifestations of anti-inflammatory responses that appear in association with systemic inflammatory response syndrome (SIRS)-like reactions

In association with the systemic inflammatory response syndrome (SIRS), anti-inflammatory response syndrome is commonly manifested in patients with trauma, burn injury, and after major surgery. These patients are increasingly susceptible to infection with various pathogens due to the excessive release of anti-inflammatory cytokines from anti-inflammatory effector cells. Recently, CC-chemokine ligand 2 (CCL2) found in the sera of mice with pancreatitis was identified as an active molecule for SIRS-associated anti-inflammatory response manifestation. Also, the inhibitory activity of glycyrrhizin (GL) on CCL2 production was reported. Therefore, the effect of GL on SIRS-associated anti-inflammatory response manifestation was investigated in a murine SIRS model. Without any stimulation, splenic T cells from mice 5 days after SIRS induction produced cytokines associated with anti-inflammatory response manifestation. However, these cytokines were not produced by splenic T cells from SIRS mice previously treated with GL. In dual-chamber transwells, IL-4-producing cells were generated from normal T cells cultured with peripheral blood polymorphonuclear neutrophils (PMN) from SIRS mice. However, IL-4-producing cells were not generated from normal T cells in transwell cultures performed with PMN from GL-treated SIRS mice. CCL2 was produced by PMN from SIRS mice, while this chemokine was not demonstrated in cultures of PMN from SIRS mice treated with GL. These results indicate that GL has the capacity to suppress SIRS-associated anti-inflammatory response manifestation through the inhibition of CCL2 production by PMN.     

Role of M2b macrophages in the acceleration of bacterial translocation and subsequent sepsis in mice exposed to whole body [137Cs] γ-irradiation

The influence of whole-body gamma-irradiation on the antibacterial host defense against Enterococcus faecalis translocation was investigated. Mice irradiated with or without 5 Gy [(137)Cs] gamma-rays were orally infected with 10(6) CFU/mouse E. faecalis. The pathogen was detected in the mesenteric lymph nodes (MLNs) of irradiated mice 1-4 d postinfection, whereas E. faecalis was not isolated from MLNs of normal mice. All irradiated mice died within 5 d of infection, whereas no mortality was shown in normal mice infected with the pathogen. Irradiated mice inoculated with normal mouse MLN macrophages (M) were shown to be resistant against the infection, although the same mice inoculated with irradiated mouse MLNM (I-MLNM) died postinfection. I-MLNM were identified as IL-10(+)IL-12(-)CCL1(+)LIGHT(+) M (M2bM) and were shown to be inhibitory on M conversion from resident M to IL-10(-)IL-12(+)M (M1M). M2bM were demonstrated in MLNs of mice 10-35 d after gamma-irradiation. M1M were not induced by E. faecalis Ag in cultures of I-MLNM, whereas normal mouse MLNM were converted to M1M in response to the Ag stimulation. After treatment with CCL1 antisense oligodeoxynucleotides, M2bM disappeared in MLNs of irradiated mice, and M1M were generated in MLNs of these mice following E. faecalis stimulation. These results indicate that M2bM presented in the I-MLNM populations were responsible for the impaired resistance of mice irradiated with gamma-rays to bacterial translocation and subsequent sepsis. E. faecalis translocation and subsequent sepsis may be controlled immunologically by the intervention of M2bM present in MLNs.     

Interleukin 12 (IL-12) is increased in tumour bearing human liver and expands CD8(+) and CD56(+) T cells in vitro but not in vivo

Human liver is enriched with CD8(+)T- and CD3(+)CD56(+) natural T (NT)-lymphocytes, important anti-tumour effectors, similar to murine NKTs. IL-12 promotes anti-tumour functions of NKTs. We quantified IL-12 and CD56(+)/CD8(+)T lymphocytes in normal and tumour bearing liver. We also examined the effect of IL-12 on the expansion/activation of peripheral blood cells in vitro. IL-12 was detected in normal (n=13, median 2032 pg/100 mg protein) and increased in tumour bearing liver (n=9, 3678 pg, p< 0.01). Infiltrating monocytes appear to be the principal producers. Culture with IL-12 selectively expanded CD8(+)T and CD3(+)CD56(+)NT cells and polarised their cytokine responses to Th1-type. However, there was no in vivo expansion of these cells in tumour bearing liver. Changes observed in culture required addition of IL-2. We therefore quantified IL-2 in hepatic tissue. IL-2 was detected in normal liver (median 4700 pg/100 mg protein). Surprisingly, there was no increase in tumour-infiltrated liver (4910 pg). The presence of IL-12 may create an environment in healthy liver that promotes the accumulation of CD8(+)T and CD56(+)NT cells. Therefore, the development of metastases in the presence of high levels of IL-12 may be due to an insufficient IL-12 response. Alternatively, lack of IL-2 rather than a defect in IL-12, may be responsible for insufficient expansion/activation of tumour specific cytotoxic T lymphocytes.