J. Di Santo

Animal models for arthritis: innovative tools for prevention and treatment

The development of novel treatments for rheumatoid arthritis (RA) requires the interplay between clinical observations and studies in animal models. Given the complex molecular pathogenesis and highly heterogeneous clinical picture of RA, there is an urgent need to dissect its multifactorial nature and to propose new strategies for preventive, early and curative treatments. Research on animal models has generated new knowledge on RA pathophysiology and aetiology and has provided highly successful paradigms for innovative drug development. Recent focus has shifted towards the discovery of novel biomarkers, with emphasis on presymptomatic and emerging stages of human RA, and towards addressing the pathophysiological mechanisms and subsequent efficacy of interventions that underlie different disease variants. Shifts in the current paradigms underlying RA pathogenesis have also led to increased demand for new (including humanised) animal models. There is therefore an urgent need to integrate the knowledge on human and animal models with the ultimate goal of creating a comprehensive ‘pathogenesis map’ that will guide alignment of existing and new animal models to the subset of disease they mimic. This requires full and standardised characterisation of all models at the genotypic, phenotypic and biomarker level, exploiting recent technological developments in ‘omics’ profiling and computational biology as well as state of the art bioimaging. Efficient integration and dissemination of information and resources as well as outreach to the public will be necessary to manage the plethora of data accumulated and to increase community awareness and support for innovative animal model research in rheumatology.

IL-2 receptor γ-chain molecule is critical for intestinal T-cell reconstitution in humanized mice

Intestinal immune cells are important in host defense, yet the determinants for human lymphoid homeostasis in the intestines are poorly understood. In contrast, lymphoid homeostasis has been studied extensively in mice, where the requirement for a functional common γ-chain molecule has been established. We hypothesized that humanized mice could offer insights into human intestinal lymphoid homeostasis if generated in a strain with an intact mouse common γ-chain molecule. To address this hypothesis, we used three mouse strains (non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) (N/S); NOD/SCID γ-chain−/− (NSG); and Rag2−/− γ-chain−/− (DKO)) and two humanization techniques (bone marrow liver thymus (BLT) and human CD34+ cell bone marrow transplant of newborn mice (hu)) to generate four common types of humanized mice: N/S-BLT, NSG-BLT, NSG-hu, and DKO-hu mice. The highest levels of intestinal human T cells throughout the small and large intestines were observed in N/S-BLT mice, which have an intact common γ-chain molecule. Furthermore, the small intestine lamina propria T-cell populations of N/S-BLT mice exhibit a human intestine-specific surface phenotype. Thus, the extensive intestinal immune reconstitution of N/S-BLT mice was both quantitatively and qualitatively better when compared with the other models tested such that N/S-BLT mice are well suited for the analysis of human intestinal lymphocyte trafficking and human-specific diseases affecting the intestines.

Heat Shock Treatment Increases Engraftment of Transplanted Human Myoblasts Into Immunodeficient Mice

Myoblast transfer therapy (MTT) is a strategy that has been proposed to treat some striated muscle pathologies. However, the first therapeutic trials using this technique were unsuccessful due to the limited migration and early cell death of the injected myoblasts. Various strategies have been considered to increase myoblast survival in the host muscle after MTT. Overexpression of heat shock proteins (HSPs) in mouse myoblasts has been shown to improve cell resistance against apoptosis in vitro and in vivo. Our objective was to determine whether heat shock (HS) treatment increased the survival of human myoblasts leading to better participation of the injected cells in muscle regeneration. For this study, HS-treated human myoblasts were injected into the tibialis anterior (TA) muscles of immunodeficient RAG-/- gammaC-/- mice. TA muscles were excised at 24 hour and at 1 month after injection. Our results showed that HS treatment increased the expression of the hsp70 protein and protected the cells from apoptosis in vitro. HS treatment dramatically increased the number of human fibers present at 1 month after injection when compared with nontreated cells. Interestingly, HS treatment decreased apoptosis at 24 hour after human myoblast injection, but no differences were observed concerning proliferation, suggesting that the increased fiber formation among the HS-treated group was probably due to decreased cell death. These data suggested that HS treatment might be used in the clinical context to improve the success of MTT.

Bacterial virulence factor inhibits caspase-4/11 activation in intestinal epithelial cells

The human pathogen enteropathogenic Escherichia coli (EPEC), as well as the mouse pathogen Citrobacter rodentium, colonize the gut mucosa via attaching and effacing lesion formation and cause diarrheal diseases. EPEC and C. rodentium type III secretion system (T3SS) effectors repress innate immune responses and infiltration of immune cells. Inflammatory caspases such as caspase-1 and caspase-4/11 are crucial mediators of host defense and inflammation in the gut via their ability to process cytokines such as interleukin (IL)-1β and IL-18. Here we report that the effector NleF binds the catalytic domain of caspase-4 and inhibits its proteolytic activity. Following infection of intestinal epithelial cells (IECs) EPEC inhibited caspase-4 and IL-18 processing in an NleF-dependent manner. Depletion of caspase-4 in IECs prevented the secretion of mature IL-18 in response to infection with EPECΔnleF. NleF-dependent inhibition of caspase-11 in colons of mice prevented IL-18 secretion and neutrophil influx at early stages of C. rodentium infection. Neither wild-type C. rodentium nor C. rodentiumΔnleF triggered neutrophil infiltration or IL-18 secretion in Cas11 or Casp1/11-deficient mice. Thus, IECs have a key role in modulating early innate immune responses in the gut via a caspase-4/11—IL-18 axis, which is targeted by virulence factors encoded by enteric pathogens.

Humanized Immune System (HIS) Mice as a Tool to Study Human NK Cell Development

The study of human hematopoiesis is conditioned by access to nondiseased human tissue samples that harbor the cellular substrates for this developmental process. Technical and ethical concerns limit the availability to tissues derived from the fetal and newborn periods, while adult samples are generally restricted to peripheral blood. Access to a small animal model that faithfully recapitulates the process of human hematopoiesis would provide an important tool. Natural killer (NK) cells comprise between 10% and 15% of human peripheral blood lymphocytes and appear conserved in several species. NK cells are implicated in the recognition of pathogen-infected cells and in the clearance of certain tumor cells. In this chapter, we discuss NK cell developmental pathways and the use of humanized murine models for the study of human hematopoiesis and, in particular, human NK cell development.

Repopulation du foie de souris athymique par des hépatocytes humains fœtaux

La transplantation d’hepatocytes est une alternative interessante a la transplantation hepatique pour le traitement des maladies hepatiques severes comme l’insuffisance hepatocellulaire et les maladies metaboliques d’origine hepatique. Cette approche est limitee par le manque de donneur, par la faible capacite des hepatocytes adultes humains a proliferer in vitro et par leur mauvaise cryoconservation. De nouvelles strategies sont en cours d’etude comme l’immortalisation reversible ou l’utilisation de progeniteurs adultes. Notre approche a consiste a utiliser des hepatocytes fœtaux humains dans un modele murin. Les hepatocytes fœtaux humains ont ete isoles de foie fœtal entre 11 et 13 semaines de gestation. Ces cellules ont ete caracterisees in vitro par immunocytochimie et ont ete transplantees dans des souris athymiques par la veine porte. Ces cellules proliferent spontanement en culture et peuvent etre transduites par des vecteurs retroviraux. In vitro , 80-90 % de ces cellules sont bipotentes et expriment des marqueurs hepatocytaires (albumine, CK8 /18) et des marqueurs biliaires (CK19). Apres transplantation, ces cellules proliferent sous forme de clones morphologiquement distincts des hepatocytes murins jusqu’a representer 10 % du foie des souris. Des etudes immunohistochimiques ont montrees qu’elles expriment l’albumine humaine et qu’elles maturaient progressivement en hepatocytes adultes. Des etudes immunohistochimiques sont en cours pour evaluer leur integration et la formation de jonctions serrees avec les cellules murines. En conclusion, les hepatocytes humains fœtaux sont capables de se greffer, de proliferer et se differencier dans le foie de souris athymiques et representent une alternative interessante pour les protocoles experimentaux.