Leptospirosis is one of the most important zoonotic bacterial diseases. It is highly prevalent in the tropics, reaching over one million cases of infection per year worldwide (Costa et al., 2015; Rajapakse, 2022). Because of global climate change, heavy rains and flooding have been associated with several leptospirosis epidemics (Lau et al., 2010). Symptoms range from asymptomatic to mild febrile to severe acute infection, potentially leading to organ failure and death. Approximately 30% of cases report long-term health consequences (Costa et al., 2015; Haake and Levett, 2015).

Wild and domestic animals can be reservoir hosts, with the black rat (Rattus rattus) and the brown rat (Rattus novergicus) being the primary source of human infections (Bradley and Lockaby, 2023; Haake and Levett, 2015). Leptospira reproduces in the renal tubules of infected animals and is excreted via urine into the environment, contaminating water and soil; thus, environmental factors and sanitary conditions can favor its transmission, by indirect contact with soil and water or direct contact with infected animals (Bierque et al., 2020a; Browne et al., 2023; Desvars et al., 2011).

The innate immune response is the first barrier against bacteria. It involves pattern recognition receptors (PRRs) that interact with pathogen-associated molecular patterns (PAMPs) and activate the expression of specific genes (Cagliero et al., 2018). Toll-like receptors (TLRs) are PRRs that identify conserved microbial components, such as lipopolysaccharides (LPS). They are essential for the control of Leptospira. TLR4 is the most important LPS receptor in mice, while TLR2 is the main human receptor that responds to Leptospira LPS (Chassin et al., 2009; Fraga et al., 2011; Haake and Levett, 2015; Werts et al., 2001). TLRs trigger pro-inflammatory cascades, which activate transcription factors. As a result, cells express pro-inflammatory molecules such as cytokines, prostaglandins (PGs), nitric oxide (NO), and chemokines (Charo et al., 2019; Kawai and Akira, 2007; Santecchia et al., 2020).

Chemokines partake in the immune response against bacterial infections such as pneumonia (Standiford et al., 1996), tuberculosis (Domingo-Gonzalez et al., 2016), and leptospirosis (Silva et al., 2019). They serve as a chemoattractant, recruiting leukocytes to the site of damage/infection (Cagliero et al., 2018). CCL2 is one example of chemokine from the CC subfamily produced by many cell types, the most important of which are monocytes and macrophages (Bachelerie et al., 2014; Bose and Cho, 2013). Cytokines control the migration of monocytes and macrophages to the target tissue, providing local defense and repair of tissue damage (Deshmane et al., 2009; Uhlén et al., 2015).

Other chemokines of the CC subfamily have been extensively studied. CCL5 is a potent chemoattractant of lymphocytes and monocytes that acts via CCR1 and CCR5, contributing to the recruitment of T cells and macrophages (Araujo et al., 2018). It has been associated with cancer, atherosclerosis, inflammatory bowel diseases, and other diseases (Zeng et al., 2022). Moreover, CCL3 is produced by lymphocytes and fibroblasts, recruiting lymphocytes to sites of infection and CD8+ T cells to lymph nodes (Castellino et al., 2006). Similarly, CCL28, expressed by epithelial cells in the intestine, lung, and salivary glands, drives the migration of T and B lymphocytes to the mucosa²⁶.

The CXC subfamily of chemokines also acts as a chemoattractant for immune cells (Bachelerie et al., 2014). CXCL5 recruits neutrophils, the most abundant type of leukocyte, to sites of inflammation and contributes to the Th17 lymphocyte response (Disteldorf et al., 2015). Similarly, CXCL9 mediates lymphocytic infiltration (Tokunaga et al., 2018), and CXCL11 is correlated with CD8+ T-cell infiltration (Li et al., 2022).

There are several knowledge gaps regarding the role of chemokines in Leptospira infection. Previous findings from our group have shown that CCL2 does not interfere with the phagocytosis of Leptospira by spleen cells in either susceptible or resistant mice. However, CCL2 has been shown to have a potential to modulate other chemokines involved in the immune response (Silva et al., 2019).

Here, we report the chemokine profile found in the serum of leptospirosis patients and the potential for protein–protein interactions by STRING analysis. We found that CCL5, CXCL9, and CXCL5 were the most expressed chemokines and are probably overexpressed during infection. These findings corroborate our data showing an increase of chemokines in the spleen and lung of Balb/c (resistant strain) infected with pathogenic Leptospira (Domingos et al., 2017). It seems that these chemokines play a key role in the host defense and their resistance to a more severe Leptospira infection.

Chemokines play a major role in the host’s immune defense (Haake and Levett, 2015). Their expression is strongly related to susceptibility to leptospirosis and organ damage (Domingos et al., 2017; Silva et al., 2020). We analyzed the profile of chemokines in patients diagnosed with leptospirosis to better understand the immune response in humans. We found that CCL5 was the most expressed chemokine in humans and was upregulated over time. CCL5 promotes early protection against Leptospira spp. by preventing cytokine storms in the immune response (Vesosky et al., 2010). We have previously shown that resistant and partially resistant mouse strains show an increase in CCL5 24 h after Leptospira infection (Arent et al., 2023; Domingos et al., 2017). The CCL5 levels seen in sera from leptospirosis patients suggest that the same mechanisms occur in humans, since CCL5 is important for regulating inflammation. NK and T helper cells unable to produce CCL5 are less efficient at recruiting DCs and cytotoxic T lymphocytes (Seo et al., 2020).

Regarding CXCL9, we showed that mice susceptible to leptospirosis (HeJ) had lower levels of this chemokine when compared to resistant mice (Balb/c) (Domingos et al., 2017), suggesting that it plays an important role in the host response. CXCL9 is upregulated during infection with pathogenic Leptospira and binds to the CXCR3 receptor, which is expressed at all stages of CD4 T-cell development (Rabin et al., 2003; Shetty et al., 2021; Tannenbaum et al., 1998). The CXCR3 axis regulates the differentiation of naïve T cells into helpers 1 and drives migration to their target sites. In Salmonella infection, the CXCR3 axis controls dissemination of bacteria. In addition, the expression of CXCR3 on CD8+ T cells increases their antibacterial activity (Chami et al., 2017; Oghumu et al., 2015; Ridley and Dyer, 2022). CXCR3 has been associated with antibacterial activity against Streptococcus pyogenes, contributing to the antimicrobial protection of the gut (Egesten et al., 2007; Reid-Yu et al., 2015).

Recombinant CXCL9 has been correlated as being 10 times more potent as an antibacterial than CXCL11 and CXCL10 (Reid-Yu et al., 2015). Moreover, CCL5 and CXCL9 together have been correlated with CD8+ T-cell infiltration (Dangaj et al., 2019), indicating an important role in the control of leptospirosis.

CXCL5 is associated with neutrophil recruitment (Persson et al., 2003). The increased concentration of this molecule detected in our study in the serum of patients with leptospirosis is in line with our previous in vivo studies, which demonstrated that CXCL5 is inhibited in susceptible mice and increased in resistant ones, implying an importance for the host response (Domingos et al., 2017). In addition, in vitro experiments showed that raw macrophages treated with CCL2 had a reduction in CXCL5 expression, implying a negative correlation between them (Silva et al., 2019).

The main findings in our study were that CCL5, CXCL5, and CXCL9 are highly expressed in human leptospirosis disease. This study with human sera combined with our animal studies indicates that these chemokines play an important role in leptospirosis immunity. However, further studies are needed to clarify the role of these molecules in the effective immune response. We suggest that CCL5 could be used as a biomarker for the complementary diagnosis of the disease, since its expression was the most prominently observed in patients with leptospirosis.