Traumatic Spinal Cord Injury (SCI) is a clinical condition encompassing several causes that result in direct damage to the architecture of the nervous tissue, impairing body functions (Fehlings and Baptiste, 2005; Lima et al., 2022). Among the numerous therapeutic interventions exploring the restoration of the injured central nervous system that occurs in various conditions, including spinal cord injury models (Benowitz et al., 1999; Benowitz et al., 1999; Richter et al., 2005; Conta and Stelzner, 2008; Kumagai et al., 2013; Mitsuhara et al., 2013; Lee et al., 2014; de Almeida et al., 2015), galectins emerge as a potential target for a novel therapeutic strategy, given their implication in prognostic outcomes (Jiang et al., 2009; Mostacada et al., 2015; Prins et al., 2016; Bonsack and Sukumari-Ramesh, 2019; Venkatraman et al., 2018; Zhuang et al., 2021; Wang et al., 2023).

Galectins are a diverse group of proteins with atypical secretion pathways present in various cell types and tissues, responding to different stimuli and microenvironmental conditions. They play crucial roles in the immune system, being present in innate immune cells (dendritic cells, macrophages, mast cells, natural killer cells), adaptive immune cells (activated T and B lymphocytes), and atypical immune activation cells (yo T lymphocytes and B1 cells) (Vasta, 2009).

Spinal cord injury leads to increased galectin-3 expression near the injury site, with recruited macrophages and activated microglia aiding myelin clearance (Hausmann, 2003; Norenberg et al., 2004; Fleming et al., 2006; Stirling and Yong, 2008; Narciso et al., 2009; Rotshenker, 2009;

Shechter et al., 2009; Ren and Young, 2013; Mendonça et al., 2018).

After moderate SCI, there is an increase of galectin-3 expression across the spinal cord, as microarray analysis of different spinal cord portions distal or near the lesion epicenter has shown (Pajoohesh-Ganji et al., 2012). We have previously shown that the absence of galectin-3 influences neuroprotection and central nervous tissue regeneration following spinal cord injury in mice (Mostacada et al., 2015). In the study, we compared wild-type and knockout animals lacking the Lgals3 gene. Galectin-3-deficient animals exhibited a predominant presence of alternative phenotype (M2) macrophages seven days post-injury, associated with tissue repair and regeneration. A traumatic brain Injury model research showed increase of Arg-1 at site lesion, associated with M2 macrophage, in Galneg mice 24 in comparison with wild type mice treated with a Galectin-3 blocker (Yip et al., 2017). These animals showed significant functional improvement after 42 days and greater myelin preservation. In contrast, wild-type animals had less pronounced functional recovery linked to a predominance of classically activated (M1) macrophages producing pro-inflammatory substances that could damage surrounding tissues. These findings underscore the crucial role of galectin-3 in modulating the inflammatory and regenerative response following spinal cord injury (Jiang et al., 2009; Mostacada et al., 2015; Prins et al., 2016; Bonsack and Sukumari-Ramesh, 2019).

Throughout the SCI pathological process and the body's tissue attempts to repair the injury, chronic inflammation is often accompanied by lymphocytes abundance, which is also observed in autoimmune diseases and hypersensitivities (Costigan et al., 2009; Kumagai et al., 2013). The activation of these lymphocytes is mediated by inflammatory cytokines such as Tnfa, IL-1, and chemokines secreted by macrophages and other cells of the mononuclear phagocytic system, which assist in recruiting lymphocytes to the site of inflammation (Costigan et al., 2009; Kumagai et al., 2013). Among the activated lymphocytes, CD4+ T lymphocytes play a crucial role in amplifying the inflammatory response or controlling and suppressing the inflammatory response after lesion acute phase. One of these subtypes is the Th1 cells, which secrete the cytokine IFNy and favor the activation of classical macrophages (M1), promoting a pro-inflammatory response.

The presence of galectin-3 has been associated with proliferation of Th1 CD4+ IFNy+ cells (Boza-Serrano et al., 2014). Th17 cells, which secrete the IL-17 cytokine defends against invaders and may also play a role in autoimmune disorders, including those affecting the central nervous system diseases (Kawanokuchi et al., 2008; Shichita et al., 2009; Hill et al., 2011). Th1 CD4+ IFNy+ cells may or may not differentiate into pathogenic states depending on the cytokines present in the microenvironment (Korn et al., 2007a; Korn et al., 2007b; Korn et al., 2008; Eisenstein and Williams, 2009; Hartigan-O'Connor et al., 2011; Singh et al., 2013; Swardfager et al., 2014; Dolati et al., 2018).

Th2 cells, in turn, secrete IL-4 and IL-13, promoting the activation of alternative macrophages (M2) with anti-inflammatory characteristics and favoring the suppression of Th1 and Th17 responses, as well as stimulating a T Reg profile. These M2 macrophages are essential for tissue repair, healing, and inflammatory resolution (Sakaguchi, 2000; Battistini et al., 2003; Campbell et al., 2003; Kim et al., 2003; Johnston et al., 2003; Pace et al., 2005; Trivedi et al., 2006; Ankeny and Popovich, 2009; David and Kroner, 2011; Mostacada et al., 2015; Hu et al., 2016; Tschoe et al., 2020; Li et al., 2022).

In spinal cord injury models, B lymphocytes are present at the injured site and can produce antibodies against antigens of the central nervous system, as well as others unrelated to the SNC. These antibodies have been associated with secondary damage to nervous tissue and motor deficits (Ankeny et Popovich, 2009).

Regulatory T cells, on the other hand, modulate the inflammatory response, maintaining tolerance to autoantigens and suppressing autoimmune disorders. The suppressive action of these cells may be related to changes in other lymphocyte and macrophage profiles, including the switching from M1 to M2 profiles (Miyara et al., 2011; Burzyn et al., 2013; Chuang et al., 2016). The works of Srejovicwith Lukic (2021) and Geng et al. (2021) provide insights regarding the effect that galectin-3 has on T Reg cells. These studies suggest that galectin-3 interferes with the binding of the transmembrane protein CD147 on T Reg lymphocytes and their receptors, which negatively impacts the intracellular cascade, enabling and maintaining FOXP3 nuclear function of FOXP3 (Solstad et al., 2011). Consequently, T Reg cells become inefficient and incapable of maintaining their characteristic profile, and as a result, they shift their function towards an effector T lymphocyte profile.

In the present study, we aimed to investigate the changes in the acquired immune response after an experimental spinal cord injury in wild-type mice and galectin-3 deficiency mice. Immunohistochemical and flow cytometry analyses were conducted to verify the role of galectin-3 on T Reg, Th1, Th2, and Th17 cells in the CNS and peripheral tissues, aiming to add new information about galectin- 3 and the adaptive immune system.