Vibrio parahaemolyticus is the most common cause of bacterial, seafood-related illness in the USA. Currently, there is a dearth of published reports regarding immunity to infection with this pathogen. Here, production of both pro- and anti-inflammatory cytokines by V. parahaemolyticus-infected RAW 264.7 murine macrophages was studied. It was determined that this infection results in increased concentrations of IL-1α, IL-6, TNF-α and IL-10. Additionally, decreases in cell surface TLR2 and TLR4 and increases in T-cell co-stimulatory molecules CD40 and CD86 were discovered. The data presented here begin to identify the immune variables required to eliminate V. parahaemolyticus from infected host tissues.
cluster of differentiation
major histocompatibility complex
thermostable direct hemolysin
tumor necrosis factor-alpha
T regulatory cells
vasoactive intestinal peptide
Vibrio parahaemolyticus, a halophilic gram-negative bacillus, is the most common cause of seafood-related illness in the USA, accounting for 45% of all seafood-related infectious outbreaks . There has been a 115% increase in laboratory confirmed cases of infection with Vibrio species since 1996–1998 surveillance data were obtained, with V. parahaemolyticus accounting for 57% of all Vibrio species causing foodborne infection . Foodborne exposure, such as consumption of undercooked mollusks or crustaceans, results in a self-limiting gastroenteritis of approximately 72 hrs duration characterized by bloody or mucoid diarrhea, vomiting, nausea, fever and abdominal pain . However, individuals with chronic medical conditions such as immunodeficiencies, heart disease or diabetes are at risk of severe, life-threatening infections such as septicemia . The USA Food and Drug Administration estimates that approximately 20% of the USA population falls within this category. However, it is unknown how many of these individuals are at risk of V. parahaemolyticus exposure . Although infection with this bacterium is considered inflammatory, there is a dearth of published reports regarding immunity to this infection or characterization of the host response.
Vibrio parahaemolyticus was first isolated during a foodborne outbreak in Japan in 1950, but it was not until 1996 that the unique O3:K6 serovar emerged in Calcutta, India. Currently, serovar O3:K6 and nearly identical serovars O4:K68 and O1:KUT (untypeable) are responsible for a pandemic causing outbreaks in Asia, and North and South America during the warm summer months . Why this organism, which is TDH positive, TRH and urease negative, and possesses two type-three secretion systems, is responsible for this pandemic is not understood .
Upon exposure to bacterial pathogens such as V. parahaemolyticus, macrophages produce pro- and anti-inflammatory cytokines. Recently, Orth and colleagues reported that infection of RAW 264.7 macrophages with V. parahaemolyticus results in autophagy, whereas Calls' group reported that U937 macrophage infection induces oncosis [7, 8]. Importantly, both these findings suggest a pro-inflammatory type immune response. Furthermore, Suzuki and colleagues reported that Vibrio cholerae and Vibrio vulnificus infection of murine macrophage activates the NLRP3 inflammasome pathway, which requires NF-kB and is highly involved in the transcription of cytokines and other immune mediators . However, the mechanisms involved in V. parahaemolyticus infection remains unknown. Macrophage activation can result in both a pro- and anti-inflammatory cytokine milieu, characterized by the presence of IL-1, IL-6, IL-12, TNF-α and IL-10 . These cytokines, produced in response to TLR engagement, have roles in immune cell recruitment, modulation of co-stimulatory surface molecules and T-cell subset differentiation. By first identifying the immune processes required for efficient pathogen clearance, such as cytokine production and changes in macrophage surface marker expression, we can begin to understand how the host controls infection and achieves elimination of this organism from infected tissues. Determining the host response to V. parahaemolyticus infection will help to assist in the treatment of individuals with chronic medical conditions. Furthermore, with several murine models for studying systemic V. parahaemolyticus infection, the data presented here will begin to facilitate our understanding of immunity to infection in vivo [11-15].
Focusing on the innate immune response, human's first line of defense against pathogens, we investigated macrophage infection by assessing changes in pro- and anti-inflammatory cytokine mRNA expression, protein production and cell surface marker expression. It is well established that infection of macrophages with bacterial pathogens results in cell stimulation, resulting in cytokine production. Because V. parahaemolyticus causes gastrointestinal, systemic and wound infections, we began our investigation by utilizing the macrophage cell line RAW264.7. Our aims were to primarily establish whether V. parahaemolyticus can cause immune activation of macrophages and to determine whether this pathogen can activate macrophages in the manner described as classical activation [16, 17]. In order to determine the cytokine milieu produced in response to a V. parahaemolyticus O3:K6, RIMD 2210633 infection, we investigated both gene transcript and protein production. We assessed RAW 264.7 macrophages (TIB-71, American Type Culture Collection, Manassas, VA, USA) infected at a multiplicity of infection of 1:1 at 4 hrs post-infection for mRNA expression. Using TaqMan BHQ probes, we detected increases in several pro-inflammatory cytokines in macrophages infected with stationary phase cultures of V. parahaemolyticus as compared to uninfected controls (Table 1; Biosearch Technology, Novato, CA, USA) (ABI 7500 Fast Real-Time PCR System and Software v2.0.3, Applied Biosystems: Carlsbad, CA, USA) (Fig. 1a). Using Student's t-test, we detected a statistically significant increase (P < 0.01) when assessing mRNA transcripts for IL-1α, TNF-α, IL-23p19 and IL-6, whereas we detected no significant increase for IL-12p40 (P > 0.05). Interestingly, mRNA expression was also upregulated and significantly increased (P < 0.01) for the anti-inflammatory cytokine IL-10.
|Probe: 5′-FAM-AGGAGTTGCCAGAAAC BHQ-3′|
|IL-6||Purchased from Applied Biosystems|
To confirm the qPCR results, we measured cytokine protein production from infected macrophage cell culture supernatants using the Milliplex MAP Mouse Cytokine/Chemokine Kit (Millipore, Billerica, MA, USA) and the Luminex 100 System (Luminex, Austin, TX, USA; Fig. 1B). We determined statistically significant increases (P ≤ 0.01) in cytokine production for IL-1α and TNF-α at 4 hrs post-infection, even though there were high background concentrations of TNF-α in uninfected macrophages. Additionally, we detected significant increases for IL-1α (P < 0.05), IL-6 (P < 0.01) and IL-10 (P < 0.01) at 24 hr post-infection. Despite the presence of mRNA transcripts for IL-12p40 and IL-23p19, protein production as determined by Luminex was negative (data not shown).
Thus, we determined that infection stimulates macrophage to produce both pro- and anti-inflammatory immune responses, suggesting classical innate activation. The cytokine milieu produced during initial immune activation is essential for T-cell subset and adaptive immune response development, ultimately leading to pathogen clearance from infected host tissues. T-lymphocyte subsets recruit neutrophils to the site of inflammation; this is essential for protection against extracellular bacteria and maintenance of intestinal balance by Tregs, which control inflammation. Therefore, the cytokine profile produced in response to V. parahaemolyticus may serve to resolve infection by recruiting lymphocytes and enhancing phagocyte function, while also preventing immune-induced damage to host tissues.
In order to determine whether V. parahaemolyticus infection results in macrophage activation leading to pro-inflammatory type, immune effector cell-induced changes in surface marker expression, we utilized flow cytometry and staining of cells with fluorochrome-conjugated monoclonal antibodies. We characterized CD40 (clone 1C10), CD80 (clone 16-10A1), CD86 (clone GL1) and MHC class II (clone M5/114.15.2) co-stimulatory surface markers along with TLR2 (clone 6C2), TLR4 (clone MTS510) and CD14 (clone Sa2-8; eBioscience, San Diego, CA, USA). Analysis of uninfected compared to infected macrophage surface marker expression was performed on live cell populations only, by gating on 7-AAD-negative cells (BD Bioscience, San Diego, CA, USA). At 24 hrs post-infection, we blocked macrophage Fcγ receptors using rat IgG2b (clone 2.4G2), then performed cell staining with fluorochrome-conjugated monoclonal antibodies. We analyzed live macrophages for changes in cell surface marker expression using an Accuri C6 Flow Cytometer and CFlowPlus v.188.8.131.52 software (Accuri Cytometers, Ann Arbor, MI, USA). Infected macrophages demonstrated increased mean fluorescence for CD40 and CD86 surface expression (Fig. 2), whereas no change was evident for CD80 and MHC class II (data not shown) when compared to uninfected control cells. The increased expression of CD40 and CD86 on the surface of macrophages in response to infection with V. parahaemolyticus suggests activation of macrophage and adoption of a T cell activating phenotype, which facilitates antigen presentation and differentiation of T cells. CD4+ T cells recognize processed antigen in the context of MHC II when CD40 and CD86 are present, resulting in T cell activation, differentiation, and cytokine production. Despite an increase in CD14 detection, the mean fluorescence of TLR2 and TLR4 was decreased compared to uninfected control cells suggesting a potential immunoregulatory mechanism for preventing excessive immune activation.
In addition, we detected decreases in TLR2 and TLR4 mRNA 4 hrs post-infection (data not shown), further confirming this downward modulation. TLR2 is associated with recognition of bacterial lipoproteins, whereas TLR4 is responsible for signaling in response to the endotoxin, LPS . LPS signaling requires the association of TLR4 with its co-receptor complex, comprised of the accessory proteins CD14 and MD-2, and results in production of IL-1, IL-6 and TNF-α. Thought to prevent tissue damage following stimulation with excessive amounts of LPS, researchers have proposed several mechanisms for regulating TLR4 concentrations, including down modulation of TLR4 at the cell surface . Additionally, TLR4 decreases on the surfaces of LPS activated macrophages can be attributed to VIP, which reduces cell responses to LPS and blocks pro-inflammatory cytokine production while increasing anti-inflammatory IL-10 production [19, 20]. A mechanism causing down-modulation of TLR2 has not been clearly established.
In conclusion, we report that V. parahaemolyticus-infected macrophages produce a classical innate immune activation response characterized by pro-inflammatory cytokine gene expression for IL-1α, IL-6, IL-12p40, IL-23p19 and TNF-α, as well as IL-10, which is known for its anti-inflammatory properties. Additionally, we confirmed protein production for IL-1α, TNFα, IL-6 and IL-10. We also determined that V. parahaemolyticus infection of macrophages modulates changes in T cell co-stimulatory surface marker expression as evidenced by increased CD86 and CD40. In addition, down modulation of TLR2 and TLR4, along with production of IL-10, suggests additional immune regulatory mechanisms may be functioning to prevent damage to host tissues. Thus, our data suggest that the early immune responses of V. parahaemolyticus-infected macrophages initiate development of an adaptive response. These findings have documented mechanisms involved in early immune activation in response to V. parahaemolyticus infections and should facilitate in vivo animal studies to better define aspects of immune regulation required to combat V. parahaemolyticus infection.
This research was supported by funds from the United States Department of Agriculture, Cooperative State Research, Education, and Extension Service, National Research Initiative Grant 2008-712 35201-04535.
The authors have no conflict of interests to declare.