Magdiel Pérez-Cruz

IL-22 Defect During Streptococcus pneumoniae Infection Triggers Exacerbation of Chronic Obstructive Pulmonary Disease

Progression of chronic obstructive pulmonary disease (COPD) is linked to episodes of exacerbations caused by bacterial infections due to Streptococcus pneumoniae. Our objective was to identify during COPD, factors of susceptibility to bacterial infections among cytokine network and their role in COPD exacerbations. S. pneumoniae was used to sub-lethally challenge mice chronically exposed to air or cigarette smoke (CS) and to stimulate peripheral blood mononuclear cells (PBMC) from non-smokers, smokers and COPD patients. The immune response and the cytokine production were evaluated. Delayed clearance of the bacteria and stronger lung inflammation observed in infected CS-exposed mice were associated with an altered production of IL-17 and IL-22 by innate immune cells. This defect was related to a reduced production of IL-1β and IL-23 by antigen presenting cells. Importantly, supplementation with recombinant IL-22 restored bacterial clearance in CS-exposed mice and limited lung alteration. In contrast with non-smokers, blood NK and NKT cells from COPD patients failed to increase IL-17 and IL-22 levels in response to S. pneumoniae, in association with a defect in IL-1β and IL-23 secretion. This study identified IL-17 and IL-22 as susceptibility factors in COPD exacerbation. Therefore targeting such cytokines could represent a potent strategy to control COPD exacerbation.

Inhibition of complement component C5 protects porcine chondrocytes from xenogeneic rejection

OBJECTIVE Tissue-based xenografts such as cartilage are rejected within weeks by humoral and cellular mechanisms that preclude its clinical application in regenerative medicine. The problem could be overcome by identifying key molecules triggering rejection and the development of genetic-engineering strategies to counteract them. Accordingly, high expression of α1,2-fucosyltransferase (HT) in xenogeneic cartilage reduces the galactose α1,3-galactose (Gal) antigen and delays rejection. Yet, the role of complement activation in this setting is unknown. DESIGN To determine its contribution, we assessed the effect of inhibiting C5 complement component in α1,3-galactosyltransferase-knockout (Gal KO) mice transplanted with porcine cartilage and studied the effect of human complement on porcine articular chondrocytes (PAC). RESULTS Treatment with an anti-mouse C5 blocking antibody for 5 weeks enhanced graft survival by reducing cellular rejection. Moreover, PAC were highly resistant to complement-mediated lysis and primarily responded to human complement by releasing IL-6 and IL-8. This occurred even in the absence of anti-Gal antibody and was mediated by both C5a and C5b-9. Indeed, C5a directly triggered IL-6 and IL-8 secretion and up-regulated expression of swine leukocyte antigen I (SLA-I) and adhesion molecules on chondrocytes, all processes that enhance cellular rejection. Finally, the use of anti-human C5/C5a antibodies and/or recombinant expression of human complement regulatory molecule CD59 (hCD59) conferred protection in correspondence with their specific functions. CONCLUSIONS Our study demonstrates that complement activation contributes to rejection of xenogeneic cartilage and provides valuable information for selecting approaches for complement inhibition.

Pseudomonas aeruginosa proteolytically alters the interleukin 22-dependent lung mucosal defense.

ABSTRACT The IL-22 signaling pathway is critical for regulating mucosal defense and limiting bacterial dissemination. IL-22 is unusual among interleukins because it does not directly regulate the function of conventional immune cells, but instead targets cells at outer body barriers, such as respiratory epithelial cells. Consequently, IL-22 signaling participates in the maintenance of the lung mucosal barrier by controlling cell proliferation and tissue repair, and enhancing the production of specific chemokines and anti-microbial peptides. Pseudomonas aeruginosa is a major pathogen of ventilator-associated pneumonia and causes considerable lung tissue damage. A feature underlying the pathogenicity of this bacterium is its capacity to persist and develop in the host, particularly in the clinical context of nosocomial lung infections. We aimed to investigate the ability of P. auruginosa to disrupt immune-epithelial cells cross-talk. We found that P. aeruginosa escapes the host mucosal defenses by degrading IL-22, leading to severe inhibition of IL-22-mediated immune responses. We demonstrated in vitro that, protease IV, a type 2 secretion system-dependent serine protease, is responsible for the degradation of IL-22 by P. aeruginosa. Moreover, the major anti-proteases molecules present in the lungs were unable to inhibit protease IV enzymatic activity. In addition, tracheal aspirates of patients infected by P. aeruginosa contain protease IV activity which further results in IL-22 degradation. This so far undescribed cleavage of IL-22 by a bacterial protease is likely to be an immune-evasion strategy that contributes to P. aeruginosa-triggered respiratory infections.

Interleukin-22 protects against non-typeable Haemophilus influenzae infection: alteration during chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease is a major health problem becoming a leading cause of morbidity and mortality worldwide. A large part of these disorders is associated with acute exacerbations resulting from infection by bacteria, such as non-typeable Haemophilus influenzae (NTHi). Our understanding of the pathogenesis of these exacerbations is still elusive. We demonstrate herein that NTHi infection of mice chronically exposed to cigarette smoke (CS), an experimental model of chronic obstructive pulmonary disease (COPD), not only causes acute pulmonary inflammation but also impairs the production of interleukin (IL)-22, a cytokine with potential anti-bacterial activities. We also report that mice lacking IL-22, as well as mice exposed to CS, have a delayed clearance of NTHi bacteria and display enhanced alveolar wall thickening and airway remodeling compared with controls. Supplementation with IL-22 not only boosted bacterial clearance and the production of anti-microbial peptides but also limited lung damages induced by infection both in IL-22−/− and CS-exposed mice. In vitro exposure to CS extract altered the NTHi-induced IL-22 production by spleen cells. This study shows for the first time that a defect in IL-22 is involved in the acute exacerbation induced by NTHi infection during experimental COPD and opens the way to innovative therapeutic strategies.

T cells expressing chimeric antigen receptor promote immune tolerance

Cellular therapies based on permanent genetic modification of conventional T cells have emerged as a promising strategy for cancer. However, it remains unknown if modification of T cell subsets, such as Tregs, could be useful in other settings, such as allograft transplantation. Here, we use a modular system based on a chimeric antigen receptor (CAR) that binds covalently modified mAbs to control Treg activation in vivo. Transient expression of this mAb-directed CAR (mAbCAR) in Tregs permitted Treg targeting to specific tissue sites and mitigated allograft responses, such as graft-versus-host disease. mAbCAR Tregs targeted to MHC class I proteins on allografts prolonged islet allograft survival and also prolonged the survival of secondary skin grafts specifically matched to the original islet allograft. Thus, transient genetic modification to produce mAbCAR T cells led to durable immune modulation, suggesting therapeutic targeting strategies for controlling alloreactivity in settings such as organ or tissue transplantation.

Boosted Rat Natural Xenoantibodies Cross-React with Enterococcus faecalis by Targeting Melibiose and L-Rhamnose

Natural antibodies include a subset described as xenoantibodies considered to be directed at microorganisms and also cross-react with antigens of unrelated species. In this study, we generated T-cell-independent (TI) and T-cell-dependent (TD) xenoantibodies in Lewis rats with hamster and pig blood injections. TI anti-hamster and anti-pig IgM and IgG xenoantibodies cross-reacted with Enterococcus faecalis but not with Escherichia coli isolated from the blood of Lewis rats after cecal ligation and puncture (CLP). TI anti-pig IgM xenoantibodies also showed some reactivity with two human blood isolates of E. faecalis. In contrast, TD xenoantibodies did not show any reactivity with rat or human bacteria. TI and TD anti-hamster and anti-pig IgM and IgG xenoantibodies showed cross-reactivity with lymphocytes and endothelial cells from species distinct to that used for immunization. Glycan array analysis and inhibition assays identified antibodies against melibiose and L-rhamnose as mediators of anti-hamster and anti-porcine xenoantibody cross-reactivity with E. faecalis. A rise in TI anti-hamster and anti-pig xenoantibodies was accompanied by decreased survival of Lewis rats in a low-severity sepsis model of CLP. Therefore, TI xenoantibodies in the rat include anti-carbohydrate antibodies reactive to bacteria of endogenous flora. Enhancement of these antibodies may result in more severe infectious diseases caused by these microorganisms.

Cellular studies for in vitro modeling of xenogeneic immune responses.

Cellular studies are essential in the xenotransplantation field in order to investigate the cellular immune responses triggered by xenogeneic cells and identify the key molecules involved. A series of functional studies can be conducted with this purpose that include treatment with proinflammatory cytokines and xenogeneic cell-based assays that put together pig cells and human leukocytes such as monocytes, NK cells, and T cells. The choice of the pig cell type is critical to appropriately model the transplant setting of interest. Thus, pig endothelial cells are commonly used for studying the rejection process of vascularized organs. Treatment with cytokines allows studying the regulation of adhesion, costimulatory molecules, and receptors involved in triggering the immune response in an attempt to reproduce the more complex in vivo situation. The adhesion assays are used to determine the capacity of human leukocytes to adhere to porcine cells under various conditions. Furthermore, we describe coculture, costimulatory, and cytotoxicity assays for investigating the cellular and molecular mechanisms that take place during the xenogeneic immune response.

Inactivation of the interleukin-22 pathway in the airways of cystic fibrosis patients

&NA; Interleukin (IL)‐22 plays a critical role in regulating the maintenance of the mucosal barrier. As airway epithelial regeneration is abnormal in cystic fibrosis (CF), we investigated IL‐22 integrity in CF. We first demonstrated, using Il‐22‐/‐ mice, that IL‐22 is important to prevent lung damage induced by the CF pathogen Pseudomonas aeruginosa. Next, IL‐22 receptor was found normally expressed at the airway epithelial surfaces of CF patients. In wound‐healing assays, IL‐22‐treated CF cultures had higher wound‐closure rate than controls, suggesting that IL‐22 signaling per se could be functional in a CF context. However, persistence of neutrophil‐derived serine‐proteases is a major feature of CF airways. Remarkably, IL‐22 was found altered in this protease‐rich inflammatory microenvironment; the serine protease‐3 being the most prone to fully degrade IL‐22. Consequently, we suspect an acquired deficiency of the IL‐22 pathway in the lungs of CF patients due to IL‐22 cleavage by the surrounding neutrophil serine‐proteases.

Biodistribution and Immunogenicity of Allogeneic Mesenchymal Stem Cells in a Rat Model of Intraarticular Chondrocyte Xenotransplantation

Xenogeneic chondrocytes and allogeneic mesenchymal stem cells (MSC) are considered a potential source of cells for articular cartilage repair. We here assessed the immune response triggered by xenogeneic chondrocytes when injected intraarticularly, as well as the immunoregulatory effect of allogeneic bone marrow-derived MSC after systemic administration. To this end, a discordant xenotransplantation model was established by injecting three million porcine articular chondrocytes (PAC) into the femorotibial joint of Lewis rats and monitoring the immune response. First, the fate of MSC injected using various routes was monitored in an in vivo imaging system. The biodistribution revealed a dependency on the injection route with MSC injected intravenously (i.v.) succumbing early after 24 h and MSC injected intraperitoneally (i.p.) lasting locally for at least 5 days. Importantly, no migration of MSC to the joint was detected in rats previously injected with PAC. MSC were then administered either i.v. 1 week before PAC injection or i.p. 3 weeks after to assess their immunomodulatory function on humoral and adaptive immune parameters. Anti-PAC IgM and IgG responses were detected in all PAC-injected rats with a peak at week 2 postinjection and reactivity remaining above baseline levels by week 18. IgG2a and IgG2b were the predominant and long-lasting IgG subtypes. By contrast, no anti-MSC antibody response was detected in the cohort injected with MSC only, but infusion of MSC before PAC injection temporarily augmented the anti-PAC antibody response. Consistent with a cellular immune response to PAC in PAC-injected rats, cytokine/chemokine profiling in serum by antibody array revealed a distinct pattern relative to controls characterized by elevation of multiple markers at week 2, as well as increases in proliferation in draining lymph nodes. Notably, systemic administration of allogeneic MSC under the described conditions did not diminish the immune response. IL-2 measurements in cocultures of rat peripheral blood lymphocytes with PAC indicated that PAC injection induced some T-cell hyporesponsiveness that was not enhanced in the cohorts additionally receiving MSC. Thus, PAC injected intraarticularly in Lewis rats induced a cellular and humoral immune response that was not counteracted by the systemic administration of allogeneic MSC under the described conditions.

Divergence of the response induced by xenogenic immunization in the sepsis survival of rats.

We have previously described that boosted natural xenoantibodies in rats cross-react to bacteria by targeting carbohydrate antigens. This type of immunization is associated with reduced survival after cecal ligation and puncture (CLP). In the present study, we investigated further this phenomenon by immunizing Lewis rats with three intraperitoneal injections, every other day, of hamster blood compared to saline-injected control animals. One day after the last injection, CLP was performed to produce a low-grade sepsis. Induction of xenoantibodies was associated with a reduction in animal survival after CLP relative to controls (45% vs. 90%, p<0.01). No bacterial blood load was observed after CLP in this model either with or without xenoantibody enhancement, indicating that the augmented mortality was not mediated by a direct effect of boosted xenoantibodies over blood bacteria. Nevertheless, the xenoimmunization produced a systemic inflammatory response in all rats. Additionally, a lack of weight gain at the time of CLP was present in animals that died after the procedure, which was not observed in surviving rats and controls. The cytokine profile at the time of CLP in animals that died after the procedure was characterized by an increase in the serum level of several cytokines, particularly adipokines. In contrast, the cytokine profile at CLP of xenoimmunized rats that survived the procedure was characterized by a reduction in the level of cytokines. In conclusion, this study failed to show a direct effect of boosted xenoantibodies over blood bacterial isolates as cause for the decreased survival after CLP. However, it evidenced that non-infectious systemic inflammation may lead to a pattern of augmented cytokines, particularly adipokines, which impairs survival after subsequent CLP. Therefore, the profile of cytokines existing before the infectious insult appears more crucial than that resulting from the condition for the outcome of sepsis.