MicroRNA-155 expression with Brucella infection in vitro and in vivo and decreased serum levels of MicroRNA-155 in patients with brucellosis

Infection with Brucella is characterized by the inhibition of host immune responses. MicroRNA-155 (miR-155) has been implicated in the immune response to many diseases. In this study, its expression during Brucella 16M infection of macrophages and mice was analyzed. Expression of miR-155 was significantly induced in macrophages at 24 h post infection. Further, an analysis of infected mice showed that miR-155 was inhibited at 7 and 14 days but induced at 28 days. Interestingly, this trend in induction or inhibition was reversed at 7 and 14 days in 16M△virB-infected mice. This suggested that decreased expression of miR-155 at an early stage of infection was dependent on intracellular replication. In humans with brucellosis, serum levels of miR-155 were significantly decreased compared to those in individuals without brucellosis and healthy volunteers. Significant correlations were observed between serum level of miR-155 and serum anti-Brucella antibody titers and the sweating symptom. This effect suggests that Brucella interferes with miR-155-regulated immune responses via a unique mechanism. Taken together, data from this study indicate that Brucella infection affects miR-155 expression and that human brucellosis patients show decreased serum levels of miR-155.

Brucellosis is a zoonosis found worldwide that causes great economic losses and public health problems, and there is no effective vaccine against the disease. The re-emergence of brucellosis in many countries in recent years has spurred concern over this "old disease" 1 . An outbreak of brucellosis occurred in Lanzhou, China and infected more than 3000 people in 2019. Brucella harbors a set of virulence effectors that hijack host cells to facilitate its own survival and replication. It uses stealth mechanisms to avoid inducing a significant immune response. These characteristics of Brucella make it a successful intracellular pathogen 2 . Human brucellosis is characterized by atypical symptoms, including fever, sweating, arthralgia/arthritis, and other constitutional symptoms, as well as hepatomegaly and splenomegaly 3 . Brucellosis is often misdiagnosed or its diagnosis is delayed, resulting in chronic infections that are difficult to cure. Nucleic acid detection can be used for early diagnosis and can increase the diagnosis window 4 . However, the low concentration of Brucella in clinical samples and inconsistency in levels of serum antibody and bacterial DNA make it difficult to evaluate the diagnostic and prognostic performance of nucleic acid-based assays. Therefore, biomarkers would be of great value for diagnosis and to determine the prognosis of brucellosis.
MicroRNAs (miRNA) are endogenous 22-nucleotide RNAs that play important gene regulatory roles. As a class of small non-coding RNAs, they are highly conserved across various eukaryotic species and function as key regulators of gene expression at the post-transcriptional level by targeting mRNAs for translational repression or degradation 5 . MiRNAs also modulate innate and adaptive immune responses to pathogens. The application of miRNAs as diagnostic or prognostic biomarkers has been demonstrated for various diseases 6  www.nature.com/scientificreports/ to their well-known role in cancer, the role of miRNAs in susceptibility and resistance to infectious diseases, and especially those of bacterial origin, is still poorly understood. Several miRNAs have been reported to finetune innate and adaptive immune responses to mycobacterial infection [7][8][9][10] . Brucella infection is characterized by a weak immune response, which can be attributed to its immune evasion-strategy. The correlation between Brucella infection and miRNA expression remains largely unknown. A recent study showed that the infection of macrophage RAW264.7 cells with Brucella significantly altered miRNA expression profiles, suggesting that miRNAs are involved in interactions between Brucella and its hosts 11 . The differences in miRNA expression patterns among human patients, however, remain to be evaluated. MiR-155 plays a central role in immune responses, and particularly in innate immunity 12 . More specifically, miR-155 is known to regulate immune responses to various infections. Mycobacterium tuberculosis infection was previously found to significantly induce miR-155 expression 13 , and miR-155 was shown to promote autophagy to eliminate intracellular M. tuberculosis 14 . One study shows that staphylococcal enterotoxins induce the expression of the oncogenic microRNA miR-155 in primary malignant T cells 15 . And upregulation of Mir-155 in RAW264.7 macrophages after Salmonella infection enhances cell death due to necroptosis by targeting RIP1/3 and Poly (ADP-ribose) polymerase-1 (PARP-1) 16 . However, whether miR-155 is involved in the immune response to Brucella infection remains largely unknown. To probe the possible roles of miR-155 in Brucella infection, in the present study, we examined miR-155 expression during Brucella infection of macrophages and mice and analyzed serum levels of miR-155 in patients with brucellosis.

Results
Expression levels of miR-155 in macrophages are altered by Brucella infection. To test whether the expression of miR-155 could be affected by Brucella infection, mouse RAW 264 macrophages and human THP-1 macrophages were infected with B. melitensis 16M or administered phosphate buffered saline (PBS). Compared with that in the uninfected PBS control group, the expression of miR-155 was significantly induced at 24 h in the 16M-infected group (Fig. 1A). The expression of miR-155 did not change significantly from 0 to 24 h post-infection (p.i.) in the PBS group, but it increased by twofold at 48 h. Compared with expression in the PBS control group, that of miR-155 was not significantly different at 0 h in the 16M group. However, the expression levels of miR-155 in the 16M group were significantly increased at 24 h and decreased at 48 h p.i., relative to levels in the controls. miR-155 expression in the 16M-infected group was increased 17-fold relative to that in the PBS group at 24 h p.i. (Fig. 1B).
Brucella wild-type strain 16M affected the expression of miR-155, suggesting that Mir-155 may be used as biomarker for diagnosis of brucellosis. Type IV secretion system (T4SS) encoded by the VirB operon is an www.nature.com/scientificreports/ important virulence factor of Brucella, whose syringe-like structure can secrete effector proteins to help Brucella escape destruction by host cells, promote Brucella replication in host cells and induce persistent infection 17,18 . Macrophage cells were also infected with the Brucella T4SS-isogenic mutant 16M△virB, and expression levels of miR-155 were compared with those induced by the wild-type strain and control conditions. Expression levels of miR-155 in the 16M△virB group were significantly increased at 0, 24, and 48 h, relative to those in the control group ( Fig. 1A, C), and expression levels with 16M△virB compared to those with 16M were increased up to 15.4-and 5.6-foldat 0 and 48 h, respectively (Fig. 1D).

Expression levels of miR-155 in mice infected with B. melitensis 16M.
To further characterize the effect of Brucella infection on miR-155, expression in Brucella-infected mice was analyzed. Spleen samples were collected from mice on 7, 14 and 28 days. At time points of 7, 14, and 28 days p.i., the relative expression levels of miR-155 in the PBS control group were 1, 0.48, and 0.31, respectively ( Fig. 2A). Compared with those in the uninfected controls, expression levels of miR-155 in the 16M-infected group were significantly decreased at 7 and 14 days. At 7 days p.i., the ratio of expression in the 16M group to that in the PBS group was decreased to 0.1, and at 14 days, this was 0.72. At 28 days, however, the ratio was increased to 1.39 (Fig. 2B). Compared with that in the PBS group, miR-155 expression in the 16M△virB group was significantly increased at day 7 and 28 ( Fig. 2A, C). At 7 days p.i., the ratio of miR-155 expression with 16M△virB to that in the 16M group was increased up to 22.6-fold, but this was decreased to 0.64-fold at 28 days (Fig. 2D). A comparison between the PBS, 16M, and 16M△virB groups showed that 16M infection resulted in a decrease in miR-155 in mice on day 7, whereas 16M△virB infection resulted in the induction of miR-155 at the same time point. This suggested that the expression level of miR-155 changed dynamically with the prolongation of Brucella infection.
Serum miRNA-155 levels are decreased in brucellosis patients. To further analyze the expression of miR-155, blood samples were collected from patients suspected of having brucellosis. After confirmation of the diagnosis, the study participants were divided into two groups, specifically brucellosis and non-brucellosis. In total, 56 patients, 47 non-brucellosis and 20 healthy volunteers, were enrolled. Demographic characteristics of the enrolled individuals were compared. No significant differences were observed in sex or age distribution. Among the enrolled patients, the most common symptom was fever (67.85%), followed by fatigue (60.71%), sweating (48.21%), joint pain (48.21%), and leg pain (44.64%) ( Table 1).
To quantify miR-155 expression, a standard curve was created using synthesized mimics. As shown in Fig. 3A, the qPCR assay could detect as little as 1 fmol/L of miR-155, and the linear curve ranged over five orders of magnitude (Fig. 3A). With this standard curve, serum concentrations of miR-155 were calculated for each sample and www.nature.com/scientificreports/ compared between brucellosis and non-brucellosis groups. Serum concentrations of miR-155 in the brucellosis group were significantly lower than those in the non-brucellosis (P < 0.001) and healthy volunteer (P < 0.001) groups (Fig. 3B)

Correlations between serum levels of miR-155 and antibody titers and clinical symptoms.
The decreased levels of miR-155 in brucellosis patients compared to those in non-brucellosis patients indicated that it might be a potential biomarker for diagnosis. This prompted us to test whether there was any correlation between the serum concentration of miR-155 and the serological assay standard agglutination test (SAT) titer and clinical symptoms. SAT antibody was tested at dilutions of 1:100 and ≥ 1:200. A significant correlation was observed between SAT titers and miR-155 levels (Fig. 4A). The mean serum level of miR-155 in the 1:100 group

Discussion
MicroRNAs play important roles in the regulation of host immune responses. miR-155, specifically, has been found to be associated with many biological processes 12,19,20 . Recent studies have also shown that miR-155 plays a role in both bacterial and viral infections 8,16,21,22 . Further, miR-155 has been shown to regulate the innate immune response, which is an important defense mechanism against invading pathogens 23 . Here, we found that the expression level of miR-155 first increased and then decreased in macrophages and increase seen in mouse spleens, whereas its expression was inhibited in human patients with brucellosis. An analysis of macrophage infections showed that miR-155 was induced at 24 h p.i. Contrary that in cell experiments, miR-155 was inhibited at days 7 and 14, but induced at day 28. Compared with that in the 16M group, the 16M△virB group showed a significant induction of miR-155. Therefore, miR-155 might be involved in responses to Brucella infection. As a key microRNA molecule, miR-155 has been studied widely. Studies have shown that it is mainly expressed in activated macrophages, dendritic cells, and B and T lymphocytes 12,19,20,24 and that miR-155 is upregulated by a variety of inflammatory mediators and pathogens. MiR-155 has been implicated in the immune responses to several bacterial pathogens, including Helicobacter pylori and M. tuberculosis 13,25 . Interestingly, miR-155 expression is enhanced by mycobacterial infection both in vivo and in vitro. Recent studies have revealed that an M. tuberculosis-purified protein-derivative highly induces the expression of miR-155 in peripheral blood mononuclear cells from patients with active tuberculosis 26 . Brucella has many characteristics that are similar to those of M. tuberculosis. Both are intracellular bacteria that cause chronic infections that are refractory to treatment. However, contrary to that observed for M. tuberculosis, miR-155 was significantly inhibited in infections of mice and patients with brucellosis. The mechanism underlying miR-155 inhibition mediated by Brucella infection remains largely unknown. However, we also observed the induction of miR-155 in macrophages at 24 h p.i. We only tested serum levels of miR-155 in this study. It will be interesting to determine whether miR-155 is also inhibited in Peripheral blood mononuclear cell (PBMC) cells from patients with brucellosis.
Brucella contains multiple virulence factors that contribute to its intracellular survival. The T4SS encoded by the virB operon is one of the most important virulence factors 18 . Inactivation of the T4SS results in reduced survival of the mutant and its inability to establish a chronic infection. Results from 16M△virB-infected mice showed that the expression of miR-155 was greatly induced. Because 16M△virB had lost its capacity to survive intracellularly and to establish a chronic infection, it can be hypothesised that the wild-type strain 16M can inhibit the induction of miR-155 and that this inhibition is essential for the establishment of chronic infection. At present, we do not know whether this inhibition of miR-155 is mediated by virB or intracellular bacterial replication. If it is mediated by virB, it is possible that the T4SS effector proteins are involved in this inhibition, because the function of the T4SS is mainly mediated by its effector proteins. Many T4SS effector proteins have been identified; however, the functions of these effector proteins remain unknown. Analyzing the roles of effector proteins in regulating miR-155-mediated immune responses will be of great value in to understand the interaction between Brucella and its hosts.
In vivo studies using miR-155-deficient mice have demonstrated that miR-155 is required for the normal immune function of T-and B-lymphocytes and dendritic cells. miR-155 has also been reported to promote the development of T helper 1 (Th1) and Th17 cell subsets and to attenuate the Th2 cell response 27 . These studies together suggest a potential role for miR-155 in the cellular immune response, which is the major arm of www.nature.com/scientificreports/ immunity that functions in anti-bacterial defenses. A Th1 response is essential for controlling Brucella. and this pathogen can interfere with host development of Th1 and Th2 responses 28 . Therefore, it is possible that miR-155 mediates host responses to Brucella. Further, Brucella can interfere with polarity in a T4SS-dependent manner. This also implies that T4SS effector might be involved in the interference with miR-155 during immune responses. The decreased serum level of miR-155 in patients with brucellosis also suggests that it could be used as an auxiliary biomarker for the diagnosis of brucellosis. Before miR-155 can be used as a biomarker, a much larger sample needs to be evaluated. Furthermore, whether there is a correlation between serum levels of miR-155 and infection stages or disease outcomes needs to be examined. With this information, it might be possible to use miR-155 as an auxiliary biomarker to monitor Brucella infection and determine disease prognosis.
In summary, in this study, we analyzed the expression of miR-155 during Brucella infection in macrophages, mice, and human patients. The results showed that miR-155 was inhibited by Brucella infection in mice. In humans, serum levels of miR-155 were decreased when compared with those in healthy volunteers. Interestingly, the inhibitory effect on miR-155 was reversed in the virB mutant, suggesting that the T4SS or intracellular survival might be involved in Brucella -mediated interference with miR-155-regulated immune responses. Further studies are needed to define this interaction between miR-155 and Brucella.  The average expression levels and standard deviations were calculated using data from two triplicates from independent experiments. Differences of expression levels in groups were measured using the Student's t-test using SPSS 20.0 with P < 0.05 considering statistically significant.

Macrophage cell and mouse infection.
Murine macrophage-like RAW264.7 cells were infected with 16M, 16M△virB, or PBS as a negative control to assess survival capability and the expression of miR-155, essentially as described previously 30 . In brief, monolayers of macrophages were seeded in 24-well plates, 1 day prior to infection, at 1 × 10 5 cells per well. Macrophages were infected with bacterial suspensions at a multiplicity of infection (MOI) of 50. At 45 min p.i post-infection, the cells were washed with PBS three times and then incubated with 50 μg/mL of gentamycin for 1 h to eliminate extra cellular bacteria. Then, replaced the cultures media were replaced with DMEM containing 20 μg/mL of gentamycin. At 0, 24, and 48 h p.i, the supernatant was discarded the cells were lysed, and the live bacteria were enumerated by plating in duplicate on TSA plates with or without kanamycin or ampicillin. Cell lysates were subject to total RNA isolation and miR-155 expression analysis. For mouse infection, groups of 10 8-week-old female BALB/c mice were intraperitoneally infected with an inoculum (2 × 10 6 CFU/mL) of 16M or 16M△virB. At 7, 14, and 28 days post-inoculation, the infected mice were sacrificed via cervical dislocation, and spleens were collected aseptically and homogenized with PBS containing 0.1% Triton X-100. Serial dilutions of spleen homogenates were prepared and plated in duplicate on TSA plates, and the CFUs were counted after 4 days of infection at 37 °C. Homogenized spleen cells were subjected to total RNA isolation and miRNA expression analysis.