Susana Brun

CXCR3, Inflammation, and Autoimmune Diseases

CXCR3 is a G protein–coupled, seven‐transmembrane receptor that binds and is activated by the three IFN‐γ‐inducible chemokines of the CXC family named CXCL9, CXCL10, and CXCL11. These chemokines are not constitutively expressed but are up‐regulated in a proinflammatory cytokine milieu. Consequently, their major function is to selectively recruit immune cells at inflammation sites, but they also play a role in angiogenesis mechanisms. In the last few years, strong experimental and clinical evidence has been obtained supporting the idea that the CXCR3 pathway is involved in the development of autoimmune diseases, especially by creating local amplification loops of inflammation in target organs, thereby inducing worsening of clinical manifestations. This article briefly reviews what we know today about the nature and functions of CXCR3, with special emphasis on its involvement in two main rheumatic systemic autoimmune diseases, namely rheumatoid arthritis and systemic lupus erythematosus.

Toll-Like Receptor Agonists Synergize with CD40L to Induce Either Proliferation or Plasma Cell Differentiation of Mouse B Cells

In a classical dogma, pathogens are sensed (via recognition of Pathogen Associated Molecular Patterns (PAMPs)) by innate immune cells that in turn activate adaptive immune cells. However, recent data showed that TLRs (Toll Like Receptors), the most characterized class of Pattern Recognition Receptors, are also expressed by adaptive immune B cells. B cells play an important role in protective immunity essentially by differentiating into antibody-secreting cells (ASC). This differentiation requires at least two signals: the recognition of an antigen by the B cell specific receptor (BCR) and a T cell co-stimulatory signal provided mainly by CD154/CD40L acting on CD40. In order to better understand interactions of innate and adaptive B cell stimulatory signals, we evaluated the outcome of combinations of TLRs, BCR and/or CD40 stimulation. For this purpose, mouse spleen B cells were activated with synthetic TLR agonists, recombinant mouse CD40L and agonist anti-BCR antibodies. As expected, TLR agonists induced mouse B cell proliferation and activation or differentiation into ASC. Interestingly, addition of CD40 signal to TLR agonists stimulated either B cell proliferation and activation (TLR3, TLR4, and TLR9) or differentiation into ASC (TLR1/2, TLR2/6, TLR4 and TLR7). Addition of a BCR signal to CD40L and either TLR3 or TLR9 agonists did not induce differentiation into ASC, which could be interpreted as an entrance into the memory pathway. In conclusion, our results suggest that PAMPs synergize with signals from adaptive immunity to regulate B lymphocyte fate during humoral immune response.

Early Differentiated CD138highMHCII+IgG+ Plasma Cells Express CXCR3 and Localize into Inflamed Kidneys of Lupus Mice

Humoral responses are central to the development of chronic autoimmune diseases such as systemic lupus erythematosus. Indeed, autoantibody deposition is responsible for tissue damage, the kidneys being one of the main target organs. As the source of pathogenic antibodies, plasma cells are therefore critical players in this harmful scenario, both at systemic and local levels. The aim of the present study was to analyze plasma cells in NZB/W lupus mice and to get a better understanding of the mechanisms underlying their involvement in the renal inflammation process. Using various techniques (i.e. flow cytometry, quantitative PCR, ELISpot), we identified and extensively characterized three plasma cell intermediates, according to their B220/CD138/MHCII expression levels. Each of these cell subsets displays specific proliferation and antibody secretion capacities. Moreover, we evidenced that the inflammation-related CXCR3 chemokine receptor is uniquely expressed by CD138highMHCII+ plasma cells, which encompass both short- and long-lived cells and mostly produce IgG (auto)antibodies. Expression of CXCR3 allows efficient chemotactic responsiveness of these cells to cognate chemokines, which production is up-regulated in the kidneys of diseased NZB/W mice. Finally, using fluorescence and electron microscopy, we demonstrated the presence of CD138+CXCR3+IgG+ cells in inflammatory areas in the kidneys, where they are very likely involved in the injury process. Thus, early differentiated CD138highMHCII+ rather than terminally differentiated CD138highMHCIIlow plasma cells may be involved in the renal inflammatory injury in lupus, due to CXCR3 expression and IgG secretion.

Autophagy in neuroinflammatory diseases

Autophagy is a metabolically-central process that is crucial in diverse areas of cell physiology. It ensures a fair balance between life and death molecular and cellular flows, and any disruption in this vital intracellular pathway can have consequences leading to major diseases such as cancer, metabolic and neurodegenerative disorders, and cardiovascular and pulmonary diseases. Recent pharmacological studies have shown evidence that small molecules and peptides able to activate or inhibit autophagy might be valuable therapeutic agents by down- or up-regulating excessive or defective autophagy, or to modulate normal autophagy to allow other drugs to repair some cell alteration or destroy some cell subsets (e.g. in the case of cancer concurrent treatments). Here, we provide an overview of neuronal autophagy and of its potential implication in some inflammatory diseases of central and peripheral nervous systems. Based on our own studies centred on a peptide called P140 that targets autophagy, we highlight the validity of autophagy processes, and in particular of chaperone-mediated autophagy, as a particularly pertinent pathway for developing novel selective therapeutic approaches for treating some neuronal diseases. Our findings with the P140 peptide support a direct cross-talk between autophagy and certain central and peripheral neuronal diseases. They also illustrate the fact that autophagy alterations are not evenly distributed across all organs and tissues of the same individual, and can evolve in different stages along the disease course.

Characterization of a new rat model for chronic inflammatory demyelinating polyneuropathies

Our objective was to develop a chronic model of EAN which could be used as a tool to test treatment strategies for CIDP. Lewis rats injected with S-palmitoylated P0(180-199) peptide developed a chronic, sometimes relapsing-remitting type of disease. Our model fulfills electrophysiological criteria of demyelination with axonal degeneration, confirmed by immunohistopathology. The late phase of the chronic disease was characterized by accumulation of IL-17(+) cells and macrophages in sciatic nerves and by high serum IL-17 levels. In conclusion, we have developed a reliable and reproducible animal model resembling CIDP that can now be used for translational drug studies.

Assessing Autophagy in Sciatic Nerves of a Rat Model that Develops Inflammatory Autoimmune Peripheral Neuropathies

The rat sciatic nerve has attracted widespread attention as an excellent model system for studying autophagy alterations in peripheral neuropathies. In our laboratory, we have developed an original rat model, which we used currently in routine novel drug screening and to evaluate treatment strategies for chronic inflammatory demyelinating polyneuropathy (CIDP) and other closely related diseases. Lewis rats injected with the S-palmitoylated P0(180-199) peptide develop a chronic, sometimes relapsing-remitting type of disease. Our model fulfills electrophysiological criteria of demyelination with axonal degeneration, confirmed by immunohistopathology and several typical features of CIDP. We have set up a series of techniques that led us to examine the failures of autophagy pathways in the sciatic nerve of these model rats and to follow the possible improvement of these defects after treatment. Based on these newly introduced methods, a novel area of investigation is now open and will allow us to more thoroughly examine important features of certain autophagy pathways occurring in sciatic nerves.

Characterization of the humoral immune response in a Lewis rat model of CIDP

Objective: Recently we have developed a reliable and reproducible preclinical chronic-EAN model in the Lewis rat that may prove useful for translational drug studies for CIDP. We have clearly shown that it is a T-cell mediated disease with an accumulation of IL-17 cells and macrophages in its late chronic phase. Since pathophysiological mechanisms involved in CIDP are believed to involve not only cellular but also humoral immunity, we therefore investigated the humoral response in our chronic-EAN model induced in the Lewis rat after injection of thiopalmitoylated P0(180–199) peptide in comparison to the classical acute EAN model induced with P0(180–199). Methods: We investigated by ELISA (at 18, 31, 43 and 57 dpi) in the sera of EAN and chronic-EAN rats the levels of antibodies directed against peptide P0(180–199), the inducing antigen. The reactivity of the antibodies obtained was tested on sciatic nerve by immunohistochemistry. To check the possibility of an epitope spreading at the late phase of the chronic disease, the reactivity of the sera at 60 dpi was tested by ELISA against different peptides described as neuritogenic: P0(56–71), P0(152–171), P0(180–199) and P2(57– 81). We also investigated the presence of B-cells in the sciatic nerve by immunohistochemistry using a mouse anti-rat CD45RA MnAb. Results: High levels of antibodies against P0(180–199) were found in chronic-EAN and EAN rats, but the antibody reactivity remained high in the chronic group for the duration of the disease, whereas it started to decline in the EAN group by day 57. At the late phase of the chronic disease (60 dpi) we found in the sera significant levels of antibodies against peptide P0(152–171) but this was not statistically different from the EAN group. No reactivity was detected against the other peptides. The rat polyclonal anti P0(180–199) antibodies labeled nicely the PNS myelin in a control sciatic nerve, highly comparable to the myelin labeling with the commercial anti-P0 MnAb. This indicates that the anti-P0(180–199) is able to recognize the corresponding peptide sequence when protein P0 is inserted in the myelin membrane. The presence of B cells was detected in the sciatic nerve of EAN rats and also of chronic-EAN rats but in greater number. Conclusion: We have indeed shown that there is a humoral response in our chronic model, but further studies are needed to clarify the roles of the antibodies and B-cells in the persistence of the chronicity of the disease.

Protective effect of 4-Phenylbutyrate against proteolipid protein mutation-induced endoplasmic reticulum stress and oligodendroglial cell death

ABSTRACT Proteolipid protein (PLP) mutation causes oligodendrocyte degeneration and myelin disorders including Pelizaeus‐Merzbacher Disease (PMD). As the pathophysiological mechanisms involved in PMD are poorly known, the development of therapies remains difficult. To elucidate the pathogenic pathways, an immortalized oligodendroglial cell line (158JP) expressing PLP mutation has been generated. Previous investigations revealed that 158JP oligodendrocytes exhibit several abnormalities including aberrant PLP insertion into the plasma membrane, cAMP, plasmalogen and cell cycle deficits. However, further clarifications of abnormal PLP‐induced oligodendrocyte degeneration are required in order to identify relevant mechanisms to target for efficient protection against oligodendrocyte death. Because PLP overexpression may lead to its accumulation inside the endoplasmic reticulum (ER) and cause ER‐stress, we explored whether ER‐stress may pivotally determine 158JP cell survival/death. Viability assays, RT‐qPCR, western blot and flow cytometry were combined to compare cell survival, ER‐stress and apoptotic markers in 158JP and control (158N) oligodendrocytes. We observed a significant decreased viability/survival of 158JP compared to 158N cells. Consistently, ER‐stress markers (BiP, caspase‐12) increased in 158JP (+30%) compared to the controls. mRNA and protein ratios of apoptotic modulators (Bax/Bcl2) are higher in 158JP oligodendrocytes which are also more vulnerable than 158N cells to tunicamycin‐induced ER‐stress. Interestingly, 4‐Phenylbutyrate (ER‐stress inhibitor), which decreased ER‐stress and apoptotic markers in 158JP cells, significantly increased their survival. Our results, which show a direct link between the viability and endogenous levels of ER‐stress and apoptotic markers in 158JP cells, also suggest that 4‐Phenylbutyrate‐based strategy may contribute to develop effective strategies against oligodendrocyte dysfunctions/death and myelin disorders. Highlights158JP oligodendrocytes harboring PLP mutation are less viable than normal 158N cells.Basal levels of ER stress and apoptosis markers are higher in 158JP than 158N cells.158JP cells are more vulnerable to tunicamycin‐evoked ER‐stress than 158N cells.ER stress pivotally determines PLP mutation‐induced 158JP oligodendrocyte death.4‐PBA which reduces ER‐stress and apoptotic levels protects 158JP cells against death.

Early differentiated plasma cells expressing CXCR3 are attracted to inflamed kidneys in lupus

Background and objectives Antibody-secreting CD138+ plasma cells are detected in the kidneys of NZB/W lupus mice where they are presumably involved in the mechanisms leading to critical renal dysfunction. Objectives of this study were to dissect the differentiation pathway of plasma cells in NZB/W mice and to understand the involvement of the identified cell intermediates in the pathogenic inflammation process in lupus. Materials and methods CD138+ cell subpopulations were identified in the spleen of NZB/W mice and further isolated by FACS. They were additionally characterised by electronic microscopy and quantitative PCR techniques. Their proliferative capacity was measured in vivo and ex vivo upon BrdU and tritiated thymidine incorporation. Antibody production was detected by ELISPOT, ELISA and intracellular flow cytometry stainings. Migrating CD138+ cells were visualised in the kidneys of diseased NZB/W mice by immunohistochemistry techniques. Results We identified and extensively characterised three cell intermediates in the differentiation pathway from B cells to plasma cells, according to their B220/CD138/MHC II expression levels. We showed that these subpopulations harbored inversely correlated proliferation and antibody secretion capacities. Very interestingly, we evidenced that the inflammation-related CXCR3 chemokine receptor is preferentially expressed by early differentiated plasma cells and that these cells secrete IgG rather than IgM. Moreover, these potentially pathogenic cells are able to migrate into the inflammatory areas of NZB/W kidneys. Conclusions Early differentiated CD138 MHCII+ rather than terminally differentiated CD138+MHCII- plasma cells are involved in the renal inflammatory injury in lupus, due to CXCR3 expression and IgG secretion.