Hsp65-producing Lactococcus lactis inhibits experimental autoimmune encephalomyelitis by preventing cell migration into spinal cord

Multiple sclerosis (MS) is an immune mediated disease that affects the central nervous system of genetically predisposed individuals. Many environmental factors have been described as putative triggers of the disease onset and development. The name of the disease refers to the plaques formed by hardened scar tissue (sclerosis) observed diffusely in the affected organs [1]. Pathological changes caused by multiple sclerosis can affect brain, spinal cord and the optic nerves [2]. MS is the leading cause of non-traumatic neurological disability in young adults in Europe as well as in the United States and its prevalence is growing worldwide. The first manifestations usually begin between 20 and 30 years, with the development of the disease being uncertain and affecting the personal and professional lives of patients [3].

Multiple sclerosis begins with an acute injury caused by CD4+ T lymphocytes that are specific for antigens of the myelin sheath. They leave the periphery and reach the central nervous system, where they are stimulated by antigen presenting cells. These lymphocytes are thought to be mostly Th1, because experimental autoimmune encephalomyelitis (EAE, the mice model of multiple sclerosis) was not induced in the absence of the p40 subunit of the cytokine IL12. Later, with the discovery of IL17-producing cells, as well as that p40 was also a common subunit of the cytokine IL23 (which acts in the maintenance of Th17 cells) [4], [5] it was shown that mice lacking IL17 or its transcription factor (RORγt) do not develop EAE either [6], [7]. Due to its high plasticity, the real mechanism and relevance of Th17 cells in some models are not easy to determine [8]. It is now known that inducing EAE by transferring Th17 cells generate a delay in disease onset and a slow progression of the disease, although clinical scores resemble the ones reached by mice that received Th1 cells. Some Th17 cells start to express IFN-γ and have a migration pattern similar to Th1 cells, which increases their pathogenic potential in the central nervous system. These cells are now known as ex-Th17 cells [9]. γδ T cells, an innate subset that does not express T cell receptor, were also found at high frequency in brains of mice with EAE. They secrete IL-17, IL-21 and IL-22, which activates Th17 cells and contribute to disease severity [10].

Circulating leukocytes also need stimulation that allow them to transpose the edge of blood vessels. This may be mediated by the selectin family of adhesion molecules and specific integrins such as α4β1 (VLA-4, very late antigen-4) that binds to VCAM-1 carbohydrate receptor (vascular cell molecule-1 adhesion), and αLβ2 (LFA-1, lymphocyte function-associated antigen-1), a receptor ligand of ICAMs (intercellular adhesion molecules [11], [12]. Furthermore, during the disease course, some chemokines and their receptors change their expression. Analysis of fluids such as blood, cerebrospinal fluid and the nervous tissue itself has showed increased levels of CXCL9 and CXCL10 chemokine and its receptor, CXCR3, which usually is detected in the majority of T lymphocytes, although this receptor can prevent cells from entering the brain parenchyma [13]. On the other hand, the receptor CCR6, ligand of CXCL20, is associated with the migration of encephalitogenic cells to the central nervous system, especially the Th17 lymphocytes [14].

Multiple sclerosis is an autoimmune disease with complex etiological, pathological, and clinical features. Treatment of MS is still a challenge, since many of the available options are not completely effective and side effects are very relevant. Therefore, the aim of many studies in this field is to reach immunoregulatory responses that allow a significant reduction of the clinical signs, without compromising the health of the individual. In this sense, oral tolerance arises as a good alternative. It consists in the modulation of the specific immune response to an antigen by its continuous oral intake [15].

Many studies have tested oral administration of antigen to induce specific tolerance and prevent autoimmune disease models [16], [17]. Our group also obtained significant protection to models of ulcerative colitis, multiple sclerosis and arthritis by using the probiotic Lactococcus lactis genetically engineered to secrete HSP65 [18], [19], [20]. These studies have used feeding protocols before disease induction that prevented it, but they did not test regimens of feeding that could interfere with ongoing autoimmune pathology.

Heat shock proteins (HSPs) are intracellular chaperones and are also very expressed in inflamed tissues, which means that they are marker molecules of cell stress [21]. The immunological factors related to HSPs are very complex. In some autoimmune disorders they act as a self-antigen and have an important immunoregulatory role since many natural Treg cells have an antigenic specificity for them. Indeed, HSP60 enhances function and survival of CD4+CD25+Foxp3+ cells [22]. Thus, HSPs may be candidates for use as antigens in oral tolerance.

In our previous work [18], we showed that HSP65-producing L.lactis prevents EAE, when it is given orally before the disease induction. This happens through an immunoregulatory mechanism, with increase of regulatory T cells in lymphoid organs (mainly CD4+Foxp3-LAP+cells, also called Th3 cells) and anti-inflammatory cytokines. Herein we focus on testing the efficacy of Hsp65-producing L.lactis during the development of experimental autoimmune encephalomyelitis (EAE) induced in C57BL/6 mice with myelin oligodendrocyte glycoprotein (MOG 35-55). The Hsp65 (a microbial 65 kDa Hsp, homologous to mammalian hsp60) was given orally for five consecutive days using the gram-positive and non-invasive bacteria Lactococcus lactis that contains a Hsp65-coding plasmid. This methodology provides the benefits of oral tolerance induced by low doses and avoids the need to isolate, purify and store this protein.

We observed an efficient reduction in the clinical and in histological scores of EAE in mice that received the L.lactis strain encoding Hsp65, and this effect occurred if the treatment started until the fourth day after disease induction. Control of EAE development was associated with increase in the frequency of regulatory LAP+ CD4+ T cells in lymphoid organs and changes in frequency of cells expressing some chemokine receptors. Homing of leucocytes to the spinal cord was also reduced in treated mice suggesting that oral tolerance induction to Hsp65 was able to decrease the entrance of encephalitogenic cells into the central nervous system. In addition, administration of HSP65-producing L.lactis 10 days before disease induction was highly efficient in preventing EAE, a result that confirms a previous study reported by our group [18]. As in the oral treatment after disease induction, we observed that fewer cells reached the spinal cord. Despite that, the frequency of regulatory T cells did not change in this organ.

In short, oral tolerance induced by Hsp65-producing L.lactis inhibited EAE development when given before or after its induction by inducing specific regulatory T cells and interfering with cellular migration of leucocytes to the spinal cord.

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