Immunomodulatory effects of bacteriocinogenic and non-bacteriocinogenic Lactococcus cremoris of aquatic origin on rainbow trout (Oncorhynchus mykiss, Walbaum)

Abstract

Lactic Acid Bacteria (LAB) are a group of bacteria frequently proposed as probiotics in aquaculture, as their administration has shown to confer positive effects on the growth, survival rate to pathogens and immunological status of the fish. In this respect, the production of antimicrobial peptides (referred to as bacteriocins) by LAB is a common trait thoroughly documented, being regarded as a key probiotic antimicrobial strategy. Although some studies have pointed to the direct immunomodulatory effects of these bacteriocins in mammals, this has been largely unexplored in fish. To this aim, in the current study, we have investigated the immunomodulatory effects of bacteriocins, by comparing the effects of a wild type nisin Z-expressing Lactococcus cremoris strain of aquatic origin to those exerted by a non-bacteriocinogenic isogenic mutant and a recombinant nisin Z, garvicin A and Q-producer multi-bacteriocinogenic strain. The transcriptional response elicited by the different strains in the rainbow trout intestinal epithelial cell line (RTgutGC) and in splenic leukocytes showed significant differences. Yet the adherence capacity to RTgutGC was similar for all strains. In splenocyte cultures, we also determined the effects of the different strains on the proliferation and survival of IgM⁺ B cells. Finally, while the different LAB elicited respiratory burst activity similarly, the bacteriocinogenic strains showed an increased ability to induce the production of nitric oxide (NO). The results obtained reveal a superior capacity of the bacteriocinogenic strains to modulate different immune functions, pointing to a direct immunomodulatory role of the bacteriocins, mainly nisin Z.

Keywords: bacteriocins; fish; immunomodulation; lactic acid bacteria; nisin; probiotics.

Figure 1

Transcriptional response of RTgutGC cells to different LAB strains: L. cremoris WA2-67 ΔnisZ, L. cremoris WA2-67 and L. cremoris WA2-67 (pJFQIAI). RTgutGC cells were incubated with 1 x 10⁶ cfu/ml of each bacterial strain at 20°C for 24 h. Subsequently, RNA was extracted and the levels of transcription of different genes analyzed by real-time PCR. Relative gene expression levels were normalized to the transcription of the housekeeping gene ef1a. Data are shown as mean fold change + SD (n = 8). The letter (a) represents transcription levels significantly different than those observed in cells not exposed to bacterial strains (control), while the asterisks indicate levels significantly different between bacterial treatments as indicated (*p ≤ 0.05 and **p ≤ 0.01).

Figure 2

Adherence of L. cremoris strains to RTgutGC cells. RTgutGC cells were incubated with the different strains at 20°C for 24 h. Control cells without bacteria were similarly treated. Adherence was then estimated by flow cytometry. Representative histograms (A) are shown along with a graph (B) representing the mean values of MFI calculated for each peak, shown as mean + SD (n = 12). The letter (a) represents adherence values significantly different to control cells.

Figure 3

Transcriptional response of splenic leukocytes to different LAB strains: L. cremoris WA2-67 ΔnisZ, L. cremoris WA2-67 and L. cremoris WA2-67 (pJFQIAI). Splenic leukocytes were incubated with 1 x 10⁶ cfu/ml of each bacterial strain at 20°C for 24 h. Subsequently, RNA was extracted and the levels of transcription of different genes were analyzed by real-time PCR. Relative gene expression levels were normalized to the transcription of the housekeeping gene bactin. Data are shown as mean fold change + SD (n = 8). The letter (a) represents transcription levels significantly different than those observed in cells not exposed to bacterial strains (control), while the asterisks indicate levels significantly different between bacterial treatments as indicated (*p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001).

Figure 4

Effects of L. cremoris WA2-67 ΔnisZ, L. cremoris WA2-67, and L. cremoris WA2-67 (pJFQIAI) strains on the percentage of splenic IgM⁺ B cells. The percentage of IgM⁺ B cells was measured via flow cytometry using a specific anti-IgM in spleen leukocyte cultures treated with 1 x 10⁶ cfu/ml of each bacterial strain for 48 h. Controls not treated with bacteria were also included. Representative dot plots (A) are included along with a graph (B) showing the percentage of live IgM⁺ B cells in the lymphocyte gate (mean + SD; n=5). The letter (a) represents transcription levels significantly different than those observed in cells not exposed to bacterial strains (control).

Figure 5

The lymphoproliferative effects of L. cremoris WA2-67 ΔnisZ, L. cremoris WA2-67 and L. cremoris WA2-67 (pJFQIAI) on B cells were determined by flow cytometry. For this, cells were stimulated with the bacteria at 20°C for 72 h, and subsequently splenic leukocytes were incubated with EdU for an additional 24 h. At that point, cells were labelled with anti-trout IgM-PE and the percentage of proliferating cells determined. Controls not treated with bacteria were also included. Representative dot plots (A) are included along with a graph (B) showing the percentage of live IgM⁺ B cells in the lymphocyte gate (mean + SD; n=9). The letter (a) represents transcription levels significantly different than those observed in cells not exposed to the bacterial strains (control), while the asterisks indicate levels significantly different between bacterial treatments as indicated (*p ≤ 0.05).

Figure 6

Effects of L. cremoris WA2-67 ΔnisZ, L. cremoris WA2-67 and L. cremoris WA2-67 (pJFQIAI) on the respiratory burst activity of splenocytes, via the reduction of the ferricytochrome c by released superoxide anion $(O_2^{-})$ . Splenocytes were incubated with the different bacterial strains for 30 min at 20°C. Optical density (OD) was then measured at 550 nm. Data are shown as mean OD values + SD (n = 9). The letter (a) represents transcription levels significantly different than those observed in cells not exposed to bacterial strains (control).

Figure 7

Capacity of L. cremoris WA2-67 ΔnisZ, L. cremoris WA2-67 and L. cremoris WA2-67 (pJFQIAI) to induce nitric oxide (NO) production in splenocytes. Cells were incubated at 20°C for 48 h with the different bacterial strains at a concentration of 1 x 10⁴ cfu/ml or with media alone. Subsequently, the optical density (OD) was measured at 540 nm and data readjusted to represent the production of NO in μM. Data are shown as mean values + SD (n = 9). The letter (a) represents NO levels significantly different than those observed in cells not exposed to bacterial strains (control), while asterisks indicate levels significantly different between bacterial treatments as indicated (*p ≤ 0.05).