Keywords
multiple sclerosis, chemokines, CXCL13, B cells, tertiary lymphoid organ, meninges
multiple sclerosis, chemokines, CXCL13, B cells, tertiary lymphoid organ, meninges
Our main goal during the writing of the revised version of this article (Version 2) entailed the clarification of concepts that should be considered as ‘hypothetical’, opposite to concepts that should be considered as facts in relation to the possible role that CXCL13 plays in the pathogenesis of MS and the possible therapeutic intervention for this disease by blocking the formation, or the effect, of this chemokine in the CNS. For this purpose, we did intensify the search for evidence of the role CXCL13 could play in the pathogenesis of EAE in the animal model of MS and the possible cellular sources of CXCL13 in the CNS, with special emphasis on the supporting findings involving the meningeal tissue. We included two new sections based on the current knowledge on the role of CXCL13 in other autoimmune diseases and the documented cellular sources of CXCL13 in neuroinflammation caused by diseases other than MS. We also included citations on the adverse effects of chemokine manipulation in the CNS of patients with MS, stressing the fact that the immune response in MS is significantly complex and not completely understood, raising flags of concern when considering extrapolation of therapeutic applications from bench to bedside. Overall, nearly 25% of the citations are new in this updated version of the manuscript.
See the authors' detailed response to the review by Hans Lassmann
See the authors' detailed response to the review by Anneli Peters
Although disease modifying therapy (DMT) agents in multiple sclerosis (MS) have contributed to reduction of neuroinflammation, they have not succeeded in the prevention of progression of disease. Inflammation is the appropriate tissue response to infection, autoimmunity, cancer, injury and allograft transplantation1. When inflammation does not resolve appropriately, a prolonged immune response persists leading to tissue destruction and loss of function1. Chronic infiltration by immune cells in the meninges is believed to form transitory lymphoid cell aggregates which simulate secondary lymphoid organs (SLO), and are known as meningeal tertiary lymphoid organs (mTLO) which play an important role in the pathogenesis of autoimmunity1,2. The mTLO seem to play a role in the intrathecal activity of immune system cells in MS3. The SLO, such as lymph nodes, show a cellular organization that includes germinal centers (GC) containing antibody secreting and proliferating B-cells with follicular dendritic cells (FDC), a T-cell zone that incorporates naïve cells from the blood stream, high endothelial venules for extravasation of lymphocytes, and a stromal cell network that provides chemokines and extracellular matrix for cell migration and structural integrity1. Chemokines are a family of proteins with the specific property of regulating leukocytes in the immune system and they may play a role in neurotransmission and neuromodulation4. Leukocyte trafficking is mediated by inflammatory chemokines in inflamed tissues and by homeostatic chemokines in lymphoid sites5 (Figure 1). In this review, we focus on the role that CXCL13 (also known as B cell attracting chemokine [BCA-1], C-X-C motif ligand 13, or B lymphocyte chemoattractant [BLC]) plays in the organization of the mTLO in MS.
The SLO have a genetically determined pattern of development and programming that allows trapping and concentration of foreign antigens to initiate an adaptive immune response1. Mucosal associated and non-encapsulated lymphoid tissue (including the Peyer’s patches, adenoid tissue of the nasopharynx, tonsils, and the bronchial associated lymphoid tissue), together with lymphoid nodes and spleen, constitute the SLO6,7. The lymph node cortex contains clusters or primary follicles that include packaged B cells and FDC, whereas the node para-cortex has a lesser number of dendritic cells (DC) and T cells6. Generation of B cells with ability to produce auto antibodies usually occur in physiological conditions8. These auto antibodies are low affinity IgM, which exhibit a wide spectrum of reactivity and strong preference for soluble self-antigens on the cell surface8. Auto reactive low affinity B cells suffer apoptosis being unlikely they represent danger in normal conditions8.
The GC present remarkable lymphocytic mitosis within SLO follicles9. Weyand et al. stated the GC are critical in the development of the B-cell normal immune response by driving-in cell division and maturation, B-cell selection with high affinity for immunoglobulin receptors and differentiation of B-cells and plasma cells (PC)2. Real time imaging technology has allowed visualization of the transit of the B cells from the dark zone to the light zone, and vice versa, during the maturation of the GC9–11. The GC light zone displays a predominance of FDC and follicular T-helper (Tfh) cells, whereas the dark zone contains closely packed lymphocytes and stromal cells9,12. The chemokine receptor CXCR4 is required for the positioning of the B cells in the dark zone where its ligand, CXCL12, is more abundant and is produced by stromal cells13. At the light zone, CXCL13 chemokine is concentrated in the FDC processes and, in conjunction with CXCR5, they contribute to the accumulation of B cells in this zone12,13. T-cells in the GC are essential to maintain signaling and represent approximately 5–20% of cell population9. Tfh cells are characterized by the expression of CXCR5 and ICOS, which has an inducible T cell co-stimulatory effect9,14. B cell growth and differentiation at the germinal center are supported by IL10 and ICOS14. Within the light zone, the three possible different outcomes for the centrocytes include death due to apoptosis; differentiation into memory B-cell or long lived plasma cells (LLPC); and re-entrance to the dark zone for a further round of cell mutation and selection15. The relevant function of the GC is, most likely, the primary production of memory B-cells and LLPC15,16 (Figure 2). Recent studies analyzing IgG heavy chain variable region genes in B cells from MS patients revealed that B cells are able to enter and exit the blood brain barrier (BBB) in order get exposed to somatic hypermutation (SMH) at the GC17–21.
The induction of lymphoid chemokines, depends on lymphotoxin β (LT-β) and the tumor necrosis factor α (TNF-α) signaling on stromal cells and FDC22. Lymphotoxin α1β2 (LTα1β2) is expressed in the surface of B and T cells in the adult immune system and ligates to the lymphotoxin β receptor (LTβR) in reticular stromal cells thus inducing expression of lymphoid chemokines, such as CCL19, CCL21 and CXCL1323. These chemokines regulate the homeostatic traffic of lymphocytes in lymphoid organs and their distribution in the GC23. Homeostatic chemokines promote secretion of LTα1β2 by T and B cells, establishing a feedback loop that perpetuates the recruitment of lymphocytes and positional organization in the GC1. The chemokine CXCL13 has the following relevant properties:
1. CXCL13 increases its own production by stimulating the growth of FDC after regulating LTα1β2 on the membrane of B cells5,26.
2. CXCL13 is produced in the SLO by FDC and macrophages and is an important chemoattractant to the CNS27,28.
3. Follicular stromal cells express CXCL13, which is needed for nesting CXCR5+B cells and a subset of T cells in the follicular compartment6.
4. CXCL13 primarily works through CXCR5 expressed in mature B lymphocytes8, CD4+ Tfh29, CD4+ Th17 cells30, minor subset of CD8+ T cells and activated tonsil Treg cells8,30.
5. CXCL13 has no relation with CD138+ and CD38+ plasmablasts, and PC17.
Stromal cells from the T cell zone express the chemokines CCL19 and CCL21, which share the receptor CCR7 that directs naïve, central memory T cells and DC to the T cell compartment6,31. CXCR5 is expressed in 20 to 30% of CD4+T cells in blood and CSF, and virtually in all B cells in blood and the majority of B cells in the CSF compartment32. Mice lacking CXCL13, or its receptor CXCR5, fail to develop peripheral lymph nodes1. Khademi et al. determined the concentration of CXCL13 in CSF of individuals with MS, other neurological diseases including viral and bacterial infection, and healthy controls finding higher levels of the chemokine in subjects with infections followed to a lesser extent by the patients with MS33. The levels of CXCL13 correlated negatively with disease span, concluding that early determination of CXCL13 might predict prognosis of disease33.
By maintaining antibody diversity, B cell differentiation, isotype switching, oligoclonal expansion, and local production of autoreactive PCs, the TLO perpetuate disease in response to environmental inputs34. Lymphoid organogenesis and formation of mTLO may be facilitated by expression of lymphotoxin α (LT-α) at the external layer of meningeal inflamed vessels leading to the compartmentalization of the immune response in MS17,35. It has been postulated that the perpetuation of neuroinflammation and disease progression results from mTLO induced differentiation and maturation of antigen specific effector lymphocytes28. The identification of independent centroblasts in the CSF of MS patients has suggested that there is an intrathecal B cell differentiation which is not dependent on the immune activity in the blood compartment17. The TLO, besides SLO, provide a thriving environment where PC differentiate from plasmablasts6,28. In the absence of recirculating immune cells from the periphery, the TLO exerts its remarkable ability to remain active for several weeks36. Therefore, the neutralization of TLO could play a significant role by blocking the re-emergency of auto reactive clones that could be able to drive relapses or resistance to therapy36. Th17 cells, Tfh and a subtype of activated B cells, which are critical in systemic inflammation related with presence of TLO, are strongly associated with MS progression37. In an animal model of experimental autoimmune encephalitis (EAE), Peters et al. found the following: 1) Expression of CXCL13 by Th17 cells in the CNS; 2) evidence that IL17 and the surface molecule podoplanin contribute to the development of ectopic lymphoid follicle in target organs; and 3) evidence of GC-like reactions in some of the mTLO as suggested by the presence of CXCL13, PNA- and GL7- positive T and B cells and plasma cells25. By using next-generation RepSeq analysis, Lehmann-Horn et al. demonstrated the presence of SHM and class switch recombination (CSR) in the mTLO B-cell aggregates which had been initiated by activation of the induced cytidine deaminase enzyme (AICDA) in the Thx2D2 mice animal model of chronic EAE38. The B-cell repertoires found at the mTLO were different than those found at the SLO suggesting that the mTLO represent autonomous sites of immune activity and that GC activity in the mTLO was antigen driven38.
Disorganized B cell follicles in SLO have shown reduced capacity to originate natural antibody responses in CXCL13-/- mice26,39. Deficiency of CXCL13 results in a moderate course of disease characterized by a better recovery with attenuation of white matter inflammation and gliosis during the acute and chronic stage of EAE40. Krumbholz et al. showed there was a direct correlation between CXCL13 levels and the number of B cells, T cells and plasmablasts in the CSF of MS patients5. Clonal expansion and SMH of B cells have been observed in the CSF of patients with MS41. CXCL13 was upregulated in active MS lesions but not in chronic inactive lesions and, in a similar range, in the serum of patients with relapsing remitting MS (RRMS) and control subjects indicating the intrathecal production of this chemokine5. CXCL13 was identified by immunohistochemistry in intrameningeal B-cell follicles, but not in the cerebral parenchyma, of chronic active or inactive MS lesions42. Patients with clinically isolated syndrome, who had shown conversion to clinically definitive MS within 2 years, had high levels of CXCL13 in the CSF33,43,44. Elevated levels of CXCL13 in CSF have also been reported in patients with RRMS compared to controls and the CSF levels have been significantly increased during relapses but declining after initiation of B cell depleting therapy22,33,45.
Chronic and active inflammation in target organs such as thymus, thyroid, synovial tissue and salivary gland can be driven by formation of TLO in the corresponding target organs2. Expression of CXCL13 in pancreatic tissue has been associated with formation of ectopic lymphoid follicles and the induction of cascade events leading to diabetes in a transgenic mice model46. In patients with Helicobacter pylori the blockade of CXCL13 has prevented the development of mucosa-associated lymphoid tissue (MALT) lymphoma and the propagation of inflammation by CXCL13 have been documented in non-lymphoid tissue47. Although formation of pulmonary lymphoid follicles has been seen in patients with complicated rheumatoid arthritis, idiopathic pulmonary hypertension and Sjogren’s syndrome, the presence of lymphoid follicles has correlated with positive outcome in patients with lung cancer and infections of the respiratory tract48. Lung B cells are a major source of CXCL13 and it has a positive role in the lymphoid neogenesis in chronic obstructive pulmonary disease through a LT receptor and toll-like receptor signaling48.
CXCL13 plays an important role in the formation of the GC in ectopic lymphoid follicles of several organs affected by inflammatory or autoimmune disease, or by infection1. Important sources of CXCL13 are follicular stromal and FDC26,49,50. In human lung tissue of patients with COPD, Litsious et al. found that stromal cells and DC produce CXCL13 upon stimulation by lymphocytes –mainly B cells- that express LT thus acquiring lymphoid tissue inducer (LTi) cells function in the TLO48. The LT stimulates the expression of CXCL13 by stromal cells mainly through the LTβ receptor in the SLO13,26,48,51. Pikor et al. reported the production of CXCL13 by meningeal stromal cells when they are in the presence of LTβR in the relapsing-remmiting model of EAE in SJL/J mice52. The source of intrathecal production of CXCL13 during neuroinflammation has not been determined with certainty. However, stromal cells within the B cell follicles have been considered to be responsible for the chemokine production and the notion of simple passive transfer from the blood stream to the intrathecal compartment due to dysfunction of the BBB has been detracted13,26,51,53. According to Essen and collaborators, stromal cells in the meninges could produce CXCL13 in special circumstances and drive the focal accumulation and organization of lymphoid cells in specific sites51. In their study, they were able to demonstrate that microglia cells, in vivo and in vitro, are the main producers of CXCL13 in acute neuroinflammation induced by the Sindbis virus, which is not associated with demyelination51. They also found that type-1 interferon could suppress the production of CXCL13 by microglial cells51. In the rhesus macaque model of neuroborreliosis, Ramesh et al. and Narayan et al. found that infiltrating microglia and macrophage/DC myeloid cells could be one source of CXCL13 in the CNS during inflammation54,55. In biopsy specimens from patients with primary CNS lymphoma, Smith and collaborators encountered the following: 1) FDC were not present in the analyzed specimens; 2) there was expression of CXCR5 and CXCL13 in malignant B cells with positive production of BCA-1 (CXCL13) mRNA; and 3) CXCL13 was present in endothelial vascular cells which had a negative production of BCA-1 (CXCL13) mRNA by in situ hybridization, a finding that could be attributed to transcytosis56. In different types of EAE, CXCL13 and BAFF mRNA transcripts were found to be significantly upregulated in the CNS of mice which developed the relapsing-remitting and the chronic-relapsing courses of disease opposite to those which developed a chronic progressive course. Besides, cells expressing CXCL13 were exclusively found in the brain stem meninges where infiltrating leukocyte proliferation was intense and vascular endothelial cells did not express CXCL1357. In specimens from patients with giant cell arteritis, arterial TLO with FDC precursors and lymphoid ducts were detected in the medial layer of the temporal arteries expressing CXCL13, BAFF, APRIL, IL 7, IL 17 and LTβ58.
Pikor et al. conducted studies in the animal model of relapsing-remitting EAE (SJL/J mice) an observed that at the onset of disease the TLO predominantly have T lymphocytes whereas in the acute an relapsing phase, the meningeal aggregates exhibited both T and B cells52. Meningeal infiltrates can be disperse or well organized encompassing mTLO, whose lifespan is unknown28,42. The presence of follicles containing proliferating B cells, T cells, PC and FDC that express CXCL13 in the proximity of inflamed blood vessels in the meninges of patients with secondary progressive MS (SPMS) has been documented42. The mTLO correlated with neuronal loss, adjacent cortical demyelination and a more rapid progression of disease22. Patients with SPMS with positive mTLO have shown wide gray matter demyelination associated with loss of neurons, oligodendrocytes, and astrocytes; cortical atrophy, and microglial activation in the outer layer of the cortex59,60. It remains to be determined whether the formation of mTLO depends on the subtype of disease or it is the result of inflammation or consequence of chronicity36.
A novel therapeutic monoclonal antibody against CXCL13 (Mab 5261 and Mab 5261-muIg) has been shown to induce functional in vitro inhibition of the chemokine in humans and mice8. An LTβ receptor blocking immunoglobulin inhibits CXCL13 interactions, suppresses the formation of mTLO in the CNS and ameliorates the symptoms of EAE in rodents23. In EAE induced by the transfer of myelin-specific Th17 cells (Th17 EAE), Quinn et al. confirmed a role of Tfh cells by blocking Tfh trafficking using antibody against CXCL13 and found that this treatment significantly reduced expression of disease61. Some DMT available for the treatment for MS ameliorate levels of CXCL13, but the mechanisms by which it occurs are not completely understood. In patients with RRMS treated with natalizumab, a significant reduction in CXCL13 in CSF was observed in comparison to β-interferon62. In another study, Novakova et al. evaluated the effect of treatment with fingolimod in CSF biomarkers, including CXCL13, of MS patients who had previously been on β-interferon, glatiramer acetate, teriflunomide (and had to switch therapy because of breakthrough disease activity) or natalizumab (who had to switch due to risk of PML) observing significant reduction of CXCL13 in the CSF of patients in both groups63. Also, Alvarez et al. found that in patients with active RRMS, in spite of treatment with β-interferon or glatiramer acetate, the administration of rituximab led to a normalization of the CSF level of CXCL13 in the majority of patients, thus suggesting that high levels of CXCL13 in CSF at baseline could predict a forthcoming therapeutic response to B cell depletion64. Piccio et al. found that in patients with RRMS treated with IV rituximab, concomitant with either β-interferon or glatiramer acetate, there was a reduction of CXCL13 and CCL19 in CSF, which correlated with significant reduction of B cells (95%) and T cells (50%) in CSF32. Perry et al. found intrathecal reduction of CXCL13 (50.4%) and IgG index (13.5%) resulting from inhibition of development of LTi cells in patients with MS treated with daclizumab65. Braendstrup et al. reported the case of a patient with MS who had undergone allogenic hematopoietic stem cells transplant for treatment of follicular lymphoma and who after two years presented negative determination of oligoclonal bands and detectable CXCL13 in CSF66. Esen et al. suggested that blockade of CXCL13 could be a possible therapeutic target in EAE with advanced state of inflammation51. However, past experience manipulating some of the chemokines in the treatment of MS has been unfavorable67,68. Atacicept is a recombinant fusion protein that ligands to the cytokines BLys/BAFF and APRIL which are involved in the differentiation, maturation and survival of B cells67,68. Although atacicept does not accomplish depletion of progenitor or memory B cells68, it has the ability to disrupt B cell pathways, thus possibly stimulating a T cell response that leads to the creation of a pro-inflammatory environment67. In a clinical trial in patients with MS, atacicept was able to reduce the concentration of immunoglobulins and the number of circulating mature B cells correlating with an increment in relapses without changes in the CNS lesion load by MRI68. Upon discontinuation of atacicept and a 60-week follow-up, laboratory findings and activity of disease normalized68. In fact, atacicept and infliximab (a TNF blocking drug) have the ability to induce increment of memory B cells in the blood, enhance their ability to enter the CNS, and increase disease relapse rate and lesion load by MRI69. Also, Badr et al. have reported evidence of synergy between BAFF and CXCL13 which could have important implications for homeostasis of B cells70. Altogether these findings have led to conclude that the immune response in MS is unpredictable and complex and that additional studies most be conducted with significant focus on patient safety67,68.
A self-sustained intrathecal inflammation fostered by CSF chemokines involved in the traffic and survival of inflammatory cells occurs early in disease and is orchestrated by mTLO3. Studies have shown that lineage of B cells can travel through peripheral blood, cervical lymphoid nodes, and the intrathecal compartment where they can be exposed to SMH in the mTLO and return to peripheral blood17. As mentioned above, Piccio et al. found that CSF CXCL13 and CCL19 were decreased at week 24 after IV rituximab32. However, Topping et al. found that therapy with intrathecal rituximab in patients with RRMS and SPMS resulted in no variation of CXCL13 levels in serum and CSF during the period of evaluation71. Bonnan has hypothesized that, in order to prevent an unwanted generalized immune suppression resulting from systemic targeting of resident TLO, intrathecal immune reset should be attempted with a combination of monoclonal antibodies targeting each cell sub-type and aimed at eliminating simultaneously B cells, T cells, PC and FDC, via the intrathecal route. Excepting rituximab, candidate drugs still require preclinical studies for validation3. Komori et al. reported that in patients with progressive MS who received therapy with intrathecal rituximab the depletion of B cells in the CSF compartment was transient and incomplete and could be facilitated by complement dependent cytotoxicity and to a lesser degree by antibody dependent celular cytotoxicity72.
An early neutralization of CXCL13 could interfere with the organization and function of the mTLO thus modifying and reducing inflammation in the CNS of patients with MS. Studies in animal models where CXCL13 has been neutralized, or is not expressed (such as the CXCL13-/- mice), confirm its crucial role maintaining, rather than initiating, inflammation and its manipulation could lead to modification of disease in these models39. However, any therapeutic strategy unable to neutralize LLPCs or antibody secreting cells will not be successful in an attempt to impede the chronic progression of disease73. Neutralization of the CXCL13 should be carefully sought as complementary therapy to the DMT in MS.
No data is associated with this article.
This work was presented, in preliminary version, at the Third annual Americas Committee for Treatment and Research in Multiple Sclerosis (ACTRIMS Forum 2018) in San Diego, CA, on February 2, 2018. Poster presentation No. P193. Financial support for the on-line publication of this article was provided by The Department of Neurology, MedStar Georgetown University Hospital.
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Competing Interests: No competing interests were disclosed.
Competing Interests: No competing interests were disclosed.
Is the topic of the review discussed comprehensively in the context of the current literature?
Yes
Are all factual statements correct and adequately supported by citations?
Partly
Is the review written in accessible language?
Yes
Are the conclusions drawn appropriate in the context of the current research literature?
Partly
References
1. Pikor NB, Astarita JL, Summers-Deluca L, Galicia G, et al.: Integration of Th17- and Lymphotoxin-Derived Signals Initiates Meningeal-Resident Stromal Cell Remodeling to Propagate Neuroinflammation.Immunity. 2015; 43 (6): 1160-73 PubMed Abstract | Publisher Full TextCompeting Interests: No competing interests were disclosed.
Is the topic of the review discussed comprehensively in the context of the current literature?
Partly
Are all factual statements correct and adequately supported by citations?
Partly
Is the review written in accessible language?
Yes
Are the conclusions drawn appropriate in the context of the current research literature?
Partly
Competing Interests: No competing interests were disclosed.
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