Inflammatory bowel diseases (IBDs), encompassing Crohn's disease (CD) and ulcerative colitis (UC), are characterized by chronic intestinal inflammation. CD, in particular, is incurable and often complicated by intestinal fibrosis, an excessive accumulation of extracellular matrix in the intestinal layers. Fibrosis affects 30-50% of CD patients, causing stenosis, fistulas, and abscesses, and accounts for 75% of surgical resections within the first decade after diagnosis. Current therapies modulate inflammation but lack effectiveness against fibrosis. Heat shock proteins (HSPs), key antigens in regulating effector T cells during inflammation, are promising antifibrotic candidates. Previous research demonstrated that HSP60 modulates immune responses, downregulating Th1 and upregulating Th2 and Treg responses, leading to increased anti-inflammatory cytokine IL-10 and reduced proinflammatory cytokines IFN-γ and TNF-α. Our group previously engineered *Lactococcus lactis* NCDO2118 to express *Mycobacterium leprae* Hsp65, preventing colitis in a murine model and ameliorating DSS-induced inflammation via Treg cell induction and IL-10/TLR2-dependent pathways. Furthermore, a new recombinant strain, *L. lactis* NCDO2118 FnBPA+(pXYCYT:Hsp65), expressing *S. aureus* fibronectin-binding protein A (FnBPA+) and *M. leprae* Hsp65, effectively reduced inflammation in acute TNBS-induced colitis. This study aimed to evaluate the anti-inflammatory and antifibrotic capacities of this invasive, Hsp65-producing strain in chronic TNBS-induced colitis.

The existing literature extensively documents the challenges posed by Crohn's disease (CD) and its frequent complication, intestinal fibrosis. Studies highlight the significant impact of fibrosis on CD patients, leading to debilitating symptoms and necessitating surgical interventions. The lack of effective antifibrotic therapies underscores the need for novel therapeutic strategies. Research on the immunomodulatory properties of heat shock proteins (HSPs), particularly their ability to regulate the balance between pro- and anti-inflammatory cytokines, has shown promise. Prior studies using various models of colitis have demonstrated the anti-inflammatory potential of HSP65 delivered by modified *Lactococcus lactis* strains. These studies provided the foundation for investigating the therapeutic potential of this approach in the context of chronic colitis and intestinal fibrosis.

Chronic colitis was induced in female BALB/c mice via intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) with increasing concentrations over six weeks, following dorsal skin presensitization with TNBS. Mice were divided into four groups (n=6 per group): (1) negative control (C−, saline i.g. and i.r.), (2) positive control (C+, saline i.g. and TNBS i.r.), (3) NFX (invasive *L. lactis* FnBPA+ without Hsp65 expression, i.g. and TNBS i.r.), and (4) NFXi (invasive *L. lactis* FnBPA+ with Hsp65 expression, i.g. and TNBS i.r.). The NFXi group received oral *L. lactis* FnBPA+ (pXYCYT:Hsp65) (1x10⁹ CFU) daily for four consecutive days per week over six weeks. Body weight was monitored weekly. On day 54, mice were euthanized, and colons were collected for histopathological evaluation (H&E and Gomori staining for inflammation and fibrosis scoring, respectively). Myeloperoxidase (MPO) activity was measured to assess inflammation. Colon homogenates were analyzed via ELISA for cytokine levels (IFN-γ, IL-12, IL-6, IL-13, IL-17, TGF-β, and IL-10). Intestinal lavage was used to measure secretory IgA (sIgA) levels. Data were analyzed using one-way ANOVA with Tukey's post-test (p<0.05).

Mice in the NFXi group (treated with Hsp65-expressing *L. lactis*) showed significantly reduced body weight loss compared to the C+ group at weeks 4 and 5, indicating a mitigation of the colitis-induced weight loss. Histological scores revealed significantly reduced inflammation and fibrosis in the NFXi group compared to the C+ and NFX groups (p<0.05). H&E staining showed less inflammatory infiltration, and Gomori staining showed decreased collagen deposition in the NFXi group. While MPO activity showed no significant difference among groups, suggesting that the impact of the treatment wasn't significant in relation to this metric, cytokine analysis revealed significantly lower IL-6 (p<0.05) and IL-13 (p<0.05) levels in the NFXi group compared to the C+ and NFX groups respectively. IL-17 levels were also significantly lower in the NFXi group. Interestingly, TGF-β levels were significantly decreased in the NFXi group compared to the NFX group. In contrast, IL-10 levels were significantly increased in the NFXi group compared to the NFX group (p<0.05), suggesting an enhanced anti-inflammatory response. sIgA levels showed no significant differences among the groups.

The findings demonstrate the therapeutic potential of orally administered Hsp65-expressing *L. lactis* in attenuating both inflammation and fibrosis in a chronic colitis model. The significant reduction in pro-fibrotic cytokines IL-13 and TGF-β, coupled with the increase in the anti-inflammatory cytokine IL-10, supports the mechanism of action. The decrease in IL-6 and IL-17, pro-inflammatory cytokines associated with Th2 and Th17 responses respectively, further underscores the anti-inflammatory effects. The lack of significant differences in MPO activity despite the histological improvements warrants further investigation. The results suggest that Hsp65 delivery by invasive *L. lactis* may shift the balance of the immune response towards an anti-inflammatory state, thereby suppressing fibrosis. This offers a promising alternative therapeutic strategy for CD.

This study demonstrates the efficacy of orally administered *L. lactis* NCDO2118 FnBPA+ (pXYCYT:Hsp65) in reducing intestinal inflammation and fibrosis in a chronic colitis model. The reduction in pro-inflammatory and pro-fibrotic cytokines, along with increased IL-10, highlights the potential of this approach for treating fibrotic CD. Future research should focus on elucidating the precise mechanisms involved and conducting clinical trials to assess the translational potential of this strategy.

This study used a murine model of chronic colitis, which may not perfectly reflect the complexity of human CD. The sample size, while sufficient for statistical analysis, could be increased to enhance the robustness of the findings. Further investigation is needed to determine the long-term effects of this treatment and to optimize the delivery method and dosage for clinical applications. The lack of significant changes in MPO activity could be due to the timing of the measurement relative to the peak inflammatory response, and additional investigation into this aspect may be needed.