Malaria and Salmonella enterica bacteremia (typhoidal or nontyphoidal) are among the most endemic diseases in the tropics (1, 2). Typhoidal serovars of Salmonella (S. typhi and S. paratyphi) are exclusively human pathogens causing bacteremic illness, whereas nontyphoidal Salmonella (NTS) serovars, such as S. enterica serovar Typhimurium (STm) and S. enteriditis, usually cause self-limiting diarrhoea with occasional secondary bacteremia (3). However, NTS serovars cause high rates of primary bacteremia in the immune-compromised host and infants in sub-Saharan Africa, often leading to conditions similar to sepsis (2).
A number of studies have reported that hemolytic malaria caused by Plasmodium species facilitates both colonization and multiplication of the Salmonella into the host. Most of the consequences about the pathogenesis of Plasmodium–Salmonella coinfection are not well known. Although, on the basis of study in animal model, a few mechanisms have been proposed, such as impairments of macrophage function (4), myeloid cell function (5), and mucosal inflammatory response (6). l-Arginine deficiency which is induced by malaria increases the susceptibility to colonization and establishment of the initial infection through an increase in the intestinal permeability and bacterial translocation associated with macrophage dysfunction (4). Another key factor is the induction of hemeoxygenase-1 (HO-1) triggered by hemolysis, which impairs oxidative burst and production of ROS in granulocytes, thus increasing the susceptibility to infection of Salmonella and also facilitating its proliferation in the host (7).
Elevated bacterial load along with malaria make the situation very hard to manage for children, immunocompromised individuals (8), and pregnant women (9).
Because of this determination of certain key factors associated with the virulence and pathophysiology, the treatment of Plasmodium–Salmonella coinfection is complicated and indispensable. In the present study, we standardized a mouse model to study the collateral observations of oxidative stress, innate immune response, pathophysiology, and the effectiveness of Ofloxacin with or without Artesunate in the condition of Plasmodium–Salmonella coinfection. Both of these drugs are recommended as effective anti-Salmonella and antimalarial for the management of typhoid fever (10) and malaria (11), respectively.
MATERIALS AND METHODS
Parasites and bacteria
Plasmodium yoelii nigeriensis167 (Pyn) was maintained in BALB/c mice (20–22 g) through regular passaging, whereas S. enterica STm (MTCC-3232) was used for the present study.
The animal study (protocol no. AH-2012-07/09) was approved by CPCSEA, Government of India through the Institutional Animal Ethics Committee. BALB/c mice were used throughout the study because these animals are genetically susceptible to lethal S. typhimurium infection (12, 13). These mice are also susceptible to lethal infection of P. yoelii nigeriensis and commonly used to study the blood stage of malaria (14).
For the standardization of the coinfection model in Balb/c mice (18–22 g), three sets (n = 6) of initial experiments were carried out through quantification of microbial load to determine the effect of Ofloxacin and standardize the model for further studies. Infection with either Pyn/STm or both was carried out on day 0 and treatments followed from day 4 to day 7 (4 doses) followed by sacrifice on day 8 for the determination of bacterial load and other parameters.
We compared the bacterial loads in spleen after oral infection of mice with STm (1 × 109 CFU of suspended in 200 μL of 5% sodium bicarbonate (15), with or without preceding Pyn coinfection (1 × 106 infected red blood cells) on day 0. After establishment of the infection, mice were treated with Olfoxacin, orally at a dose of 10, 50, and 150 mg/kg body weight for 4 days (days 4–7) to derive the ED50 of Ofloxacin in STm and Pyn coinfected mice. However, the dose of Artesunate in the coinfected groups was kept constant (80 mg/kg i.p.). The percent parasitemia was quantified through Giemsa stained thin blood smears every alternate day after Pyn infection. Mice attained peak parasitemia by day 8 (40%–50%) during which blood was collected from mice and then humanely sacrificed. Spleen was collected aseptically in normal saline solution (NSS), homogenized, diluted, and plated onto Brain Heart Infusion (BHI) Agar. After overnight incubation at 37°C, CFUs were enumerated. Hemoglobin at peak parasitemia was quantified through cyanomethemoglobin (CMG) method. Biochemical analysis of serum was done to follow the extent of liver damage in mice. Alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) activities were measured in the serum with the help of commercially available kits (Siemens, India).
The liver was also collected for histopathological studies. Briefly, after fixation of the liver sample in 10% buffered formalin and dehydration in graded concentrations of ethanol, the embedding in paraffin wax was sequentially performed. Sections (4–5 μm thick) were obtained and processed for staining with hematoxylin–eosin (H&E stain) and rendered for light microscopic examination. Microphotography of random observations was also considered. Overall, liver histopathology was classified based on the severity of four histological criteria, such as hyperplastic Kupffer cells, portal tract inflammation, sinusoidal congestion, and hemozoin deposition (16, 17). The severity level of each histopathological change was graded on a scale from 0 to 3, according to semiquantitative assessment of 5 HPF (high power field at magnification of 400×). The highest possible total score was 12 (4 histological criteria × 3 as highest scale). Score 0 meant no histopathological change and score 12 referred to most severe histopathological change. For grading of Kupffer cell hyperplasia 0 to 20, 20 to 35, 36 to 50, more than 50 cells per HPF were considered as grade 0, 1, 2, and 3, respectively. Sinusoid congestion was graded 0 to 3 as no congestion, mild congestion, moderate congestion, and severe congestion. Similarly, hemozoin deposition was graded as 0 to 3 as none, mild, moderate, or severe deposition. Portal inflammation was also graded as 0 to 3 as none, mild, moderate, and severe on the basis of the sprinkling or dense packing of the inflammatory cells in portal areas.
Measurement of oxidative stress in liver
Lipid peroxidation was determined by the reaction of malonaldehyde (MDA) with thiobarbituric acid (TBA) to form a colorimetric (532 nm)/fluorometric (λex = 532/λem = 553 nm) product, proportional to the MDA present. Briefly, 1 mL of 10% liver homogenate was prepared in phosphate buffer saline (pH 7.4), containing equal volume of 10% trichloroacetic acid (TCA) and 0.1% butylated hydroxytoluene (BHT) followed by centrifugation (13,000× g, 10 min) to remove the insoluble material. Two hundred microliter of the supernatant from each homogenized sample was placed into a microcentrifuge tube and 600 μL of TBA solution (0.67% in 50% acetic acid) was added into each vial containing standard or sample. The mixture was then incubated at 95°C for 60 min. All the tubes were cooled to room temperature in an ice bath for 10 min before recording the absorbance at 532 nm. MDA (MP Biomedicals) was used as a standard.
Measurement of GSH and GSSG
Total glutathione level in liver was determined by spectrophotometric/microplate reader assay (18). All reagents were prepared in 0.1 M potassium phosphate buffer with 5 mM EDTA disodium salt, pH 7.5 (KPE). Liver was isolated and washed in ice-cold phosphate buffer saline pH 7.4 and homogenized in a solution of 5% metaphosphoric acid and 0.6% sulfosalicylic acid mixture. The homogenate was then centrifuged at 3,000 g at 4°C for 10 min and clear supernatant was collected for the assay. Twenty microliter of supernatant from each homogenized sample was placed into a well of microtiter plate. Equal volumes of freshly prepared DTNB (Sigma, 2 mg of DTNB in 3 mL KPE) and GR (Sigma, 40 μL of GR [250 units/mL] in 3 mL KPE) solutions were added together (120 μL to each well) and allowed to stand for 30 s for the conversion of GSSG to GSH, followed by the addition of 60 μL of β-NADPH (TCI, 2 mg of β-NADPH in 3 mL KPE). The absorbance was read immediately at 412 nm in a microplate reader (SpectraMax) every 30 s for 2 min (five readings in total from 0 to 120 s). The actual total GSH concentration in the samples was derived by using linear regression to calculate the values obtained from the standard curve (ranging from 26.4 to 0.103 nM) as μM or nM/mg protein. GSH (MP Biomedicals) was used as standard.
GSSG was spectrophometrically quantified by the GSSG reductase recycling method (19). Briefly, tissue homogenates (supernatant) were treated with 2-vinylpyridine (2 μL for 100 μL of homogenate), which covalently reacts with GSH (but not GSSG). The excess 2-vinylpyridine was neutralized with triethanolamine (6 μL for each sample) for 10 min. To these derivatized samples, the procedure of GSH was repeated again to obtain the GSSG. To determine the actual total GSH and GSSG concentration in the samples by using linear regression to calculate the values obtained from the standard curve (ranging from 26.4 to 0.103 nM) as μM or nM/mg protein. GSH and GSSG (MP Biomedicals) were used as standard.
Catalase and superoxide dismutase assay
Catalase assay was carried out using liver tissue through commercial kit (Cayman Chemicals, Ann Arbor, Mich). Superoxide dismutase (SOD) was measured in liver homogenate according to method (20) based on inhibition of reduction of nitro blue tetrazolium (NBT). Liver homogenates (10%) were prepared in cold 50 mM potassium phosphate buffer (pH 7) containing 0.1 mM EDTA and 0.5% Triton X-100 and centrifuged at 12,000 g for 5 min at 4°C. Supernatant was collected and used for the assay. Assay mixture contained 120 μL sodium phosphate buffer (pH 8.3, 0.052 M), 10 μL phenazine methosulphate (TCI, 186 μM), 30 μL NBT (Sigma, 300 μM), 20 μL NADH (Sigma, 780 μM), 40 μL sample (supernatant), and 80 μL HPLC grade water. Reaction was initiated by the addition of NADH, and 30 s later the reaction was stopped by the addition of 100 μL of glacial acetic acid. Four hundred microliter of butanol was added into the reaction mixture and vortexed vigorously and allowed to stand for 10 min followed by centrifugation at 5,000 g for 5 min at 4°C. Chromogen in butanol layer was read at 560 nm against butanol in a microplate reader (SpectraMax).
Quantitative real-time PCR
Total RNA from the perfused tissues was extracted using Trizol reagent (Thermo Fisher Scientific, Waltham, Mass) according to the manufacturer's instruction. cDNA was prepared using high capacity reverse transcription kit (Thermo Fisher Scientific) and altered gene expression level compared with control was assessed by quantitative real time RT-PCR with SYBR Green as a fluorescent reporter using Maxima SYBR Green/ROX PCR Kit (Thermo Fisher Scientific) through QuantStudio 6 Flex Real Time PCR System. Beta-actin served as internal control. Calculation for relative gene expression was carried out according to the DDCt method using threshold values. Three independent experiments were performed for each group.
Detection and quantification of NFκB and HO-1
NFκB was quantified separately in cytosolic and nuclear proteins obtained from liver through western blotting. To fractionate the nuclear and cytosolic protein, liver tissues (250 mg) from different groups of mice were minced in 500 μL of hypotonic buffer pH 7.4 (20 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 1 mM PMSF; protease inhibitor cocktail; Pierce) and then homogenized. Homogenate was incubated for 15 min on ice and then added with 25 μL of 10% NP4O and vortexed for 10 s followed by centrifugation at 3,000 rpm for 10 min at 4°C. Supernatant was collected as cytoplasmic fraction and stored at −80°C. Nuclear pellet was then resuspended in 100 μL of complete lysis buffer (Sigma) containing 1 mM PMSF and protease inhibitor cocktail and incubated on ice for 30 min with intermittent vortex at each 10 min. It was centrifuged at 14,000 g for 30 min at 4°C and supernatant was collected as nuclear fraction and stored at −80°C until use.
HO-1 in the tissues and serum were detected and quantified through western blot and ELISA (Enzo Life Sciences), respectively. Briefly, protein from liver tissue was isolated through homogenization in cold tissue lysis buffer (Sigma) with protease inhibitor cocktail (1:100) and centrifuged at 13,000 rpm for 15 min. Supernatant for protein quantification through BCA method (Pierce) was denatured at 95°C on dry bath for 10 min in Laemmli buffer and loaded (100 μg) onto 10% SDS-PAGE resolving gel for separation. The separated proteins were then transferred to nitrocellulose membrane and blocked in a protein free blocking solution Advanblock-PF (Advansta) for 2 h at room temperature. Membranes were then probed overnight at 4°C with a primary antibody diluted in blocking solution. Anti HO-1 mouse monoclonal antibody (Enzo Life Sciences) and anti-NF-κB P65 rabbit polyclonal antibody (Abcam) was diluted according to manufacturer's instructions. Anti-β-actin mouse monoclonal antibody (Abcam) was used as the control. Membranes were then incubated at room temperature for 1 h. After three washes with PBS (pH 7.4) containing 0.05% (v/v) Tween 20, membranes were probed with respective horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000 in wash buffer) for 2 h at room temperature. After three washes with wash buffer, blots were visualized through Immobilon Western Chemiluminescent HRP Substrate and the relative density of bands was analyzed by Amersham Imager 600 (GE Health Care Life Sciences). Density of the bands for HO-1 and NF-κB were quantified in relation to β-actin.
Quantification of Th1/Th2 in serum
The BD CBA Mouse Th1/Th2/Th17 Cytokine Kit was used to measure interleukin-4 (IL-4), interleukin-6 (IL-6), interferon-γ (IFN-γ), tumor necrosis factor (TNF), and interleukin-10 (IL-10) protein levels in serum through flowcytometry (BD LSR II).
The significance was ascertained through one-way ANOVA and Tukey's multiple comparison test using Graphpad Prism ver 5.0. P < 0.05 was considered statistically significant (P < 0.05, P < 0.01, P < 0.001).
Malaria induces impairment of host resistance to S. typhimurium
The observations toward the standardization of the coinfection model indicated that mice infected with STm could be effectively treated with Ofloxacin at all the three doses. However, when coinfected mice (with significantly high STm load) were treated with similar doses of Ofloxacin, only the group administered with the highest dose (150 mg) responded to the treatment (Fig. 1A). In parallel, when the coinfected mice were treated with a therapeutic dose of Artesunate, even the lower doses of Ofloxacin (10 and 50 mg) were capable of reducing the microbial load (Fig. 1B). The final set of experiments clearly indicated that Ofloxacin was ineffective in reducing the bacterial load in the presence of malaria (Fig. 1C). The observations were also corroborated in terms of percent parasitemia (Fig. 1D) and hemoglobin levels (Fig. 1E). The mortality studies clearly indicated that the coinfected animals have a shortened mean survival and did not have any effect of Ofloxacin treatment in decreasing the mortality (Supplementary Fig. 1, https://links.lww.com/SHK/A694) which improved upon treatment with Artesunate alone or with the combination of Artesunate and Ofloxacin.
Liver restores its function with the clearance of parasite and bacteria
Hyperplastic Kupffer cells, portal tract inflammation, sinusoidal congestion, and hemozoin pigment deposition were important pathological hallmarks related to liver damage. Liver of uninfected mice illustrated a normal portal tract, consisting of hepatic vein and hepatic artery, with surrounding hepatocytes. Very few inflammatory cells could be observed within the portal tract (Fig. 2). The total grading score (Table 1) encompassing the histopathological grading tends to increase with the severity of the infection and was found to be highest in the coinfected group when compared with STm- or Pyn-infected mice. Ofloxacin treatment in STm-infected mice significantly reduced the grading score, but did not show any effect in the coinfected condition. In addition, the treatment of Artesunate in the coinfected mice resulted in significant reduction in the scores and was found more efficient when used in combination with Ofloxacin.
Liver damage and oxidative stress was evident with the increase in the liver enzymes (Fig. 3, A–C), elevated MDA (Fig. 3D), reduced GSH (Fig. 3E), GSSG (Fig. 3F), and catalase and SOD activity (Fig. 3, G and H). In coinfected mice this effect was additive and was thus higher than individually infected mice. Ofloxacin treatment was efficient to lower the liver enzymes and oxidative stress to a normal level in STm-infected mice, but not found effective in coinfected mice due to inability to control bacterial load in the presence of Pyn infection. Treatment of coinfected mice with either Artesunate or Artesunate–Ofloxacin combination significantly normalized all the above parameters.
TLR-2 and TLR-4 overexpression
Overexpression at m-RNA level of both TLR-2 and TLR-4 was observed in STm- and Pyn-infected mice, though the expression of TLR-2 was highly induced by Pyn and TLR-4 by STm infection (Fig. 4). Thus, in coinfected mice the expression of both TLR-2 and TLR-4 was found maximum. Expression level of TLR-2 in coinfected mice was similar to those observed in Pyn-infected mice, whereas TLR-4 expression was significantly higher than STm-infected mice due to the higher bacterial burden. Reduction in the m-RNA expression of TLR-2 and TLR-4 response was observed with the control of parasitemia and bacterial load.
The expression of Th1 (Fig. 5, A and B) and Th2 cytokines (Fig. 5, C–E) in serum was significantly enhanced in STm- and Pyn-infected mice, the highest being in coinfected mice, which reduced with the control of the parasitemia and bacteremia. Similar to the Th1 response, Th2 cytokines were also significantly increased in STm, in Pyn, and in coinfected mice. However, it did not reduce upon treatment with Ofloxacin, Artesunate, and combination of Artesunate–Ofloxacin. We also observed corroborative evidences from the mRNA expression of Th1 and Th 2 cytokines from liver tissue (Supplementary Fig. 2, https://links.lww.com/SHK/A695).
NF-κB activation and induction of HO-1
NF-κB activation was assessed through the western blotting of nuclear (Fig. 6A) and cytosolic protein (Fig. 6B) isolated and separated from liver tissue. Data indicated that there was a significant increase and decrease in NFκB level in nuclear and cytosolic proteins which directly corroborated with parasitemia and bacterial load. Activation was maximum in the coinfected group followed by Pyn- and STm-infected groups, respectively. Treatment of coinfected mice with Artesunate and the combination of Artesunate and Ofloxacin resulted in significant decrease in NFκB mobilization into the nucleus, but treatment with Ofloxacin failed to do the same.
HO-1 from serum (Fig. 6C) and tissue (Fig. 6D) was quantified through western blotting and ELISA, respectively. Results indicated an increased level of HO-1 in liver along with HO-1 concentration in serum, which was also in direct correlation with parasitemia and bacterial load. HO-1 level was significantly higher in coinfected mice followed by Pyn- and STm-infected mice, respectively. Induction of HO-1 in coinfected mice significantly reduced after treatment with Artesunate and combination of Artesunate and Ofloxacin, but not by Ofloxacin.
Clinical reports indicate that invasive bacterial disease in children infected with malaria was responsible for higher proportion of fatalities than Malaria alone and WHO criteria for antimicrobial treatment failed to contain the bacterial infection in malaria-infected subjects which is due to the higher prevalence of nontyphi Salmonella infection and its association with severe anemia (21). The treatment regimens for coinfected patients are not currently included in the WHO criteria. Furthermore, in rural areas of Africa, clinicians remain uncertain as to which patients to treat and which antimicrobial to use.
Nontyphoidal Salmonella infection is one of the most common cause of community acquired bacteremia and its coinfection has been associated with high malaria mortality (7). The present study describes the underlying responses in an animal model harboring a coinfection of P. yoelii nigeriensis and S. typhimurium along with the effect of standard treatment on various physiological and immunological parameters. In the basic model, we quantified the bacterial load on spleen and observed that the load could be significantly reduced with the dose dependant treatment of Ofloxacin. However, the increased microbial load (40–50 times) in coinfected mice (Fig. 1, A and B) is indicative of the impairment of host resistance, rendering the host susceptible to secondary infection. However, in all these separate experiments we observed that neither the presence of Salmonella nor the treatment with Ofloxacin has any effect on the progression of malaria unlike vice versa.
Clinically, both the diseases individually (22) and in coinfection lead to liver damage or hepatomegaly as evidenced by elevated liver enzymes, namely, ALP, AST, and ALT (23), with similar observations have been reported for Salmonella in rats (24). Our observations of progressing parasitemia with hemoglobin degradation resulted in deposition of free heme in the liver which might be responsible for the induction of intravascular oxidative stress due to Fe2+ reactivity (25). The combined observations of liver morphology, liver function enzymes, and the oxidative stress parameters confirm that the coinfection causes critical damage to liver which is mediated by the deposition of heme (because of malaria) and induction of inflammatory response triggered by both parasitemia and bacteremia.
We also examined the innate immune response through the expression level of Toll-like receptors (TLR-2 and TLR-4), TH1/Th2 cytokines, and NFκB. Along with regulation of various biological processes including apoptosis, stress response, immunity, and inflammation, NF-κB is also directly involved in the regulation of HO-1 (26) which is one of the key factor toward the impairment of resistance to Salmonella, resulting in the loss of efficacy of commonly used anti-Salmonella drugs at their therapeutic dose. TLRs have been shown to recognize a broad range of microbial structures (27, 28), and after activation, most TLRs induce a common intracellular signaling pathway that culminates in the activation of the nuclear factor NF-кB. This key transcription factor in turn activates the expression of chemokines, cytokines, and cell–surface molecules such as adhesions, selectins, integrins, and costimulatory molecules (27–29). A substantial amount of data suggests that TLR4 is involved mainly in the recognition of LPS from gram-negative bacteria (29–31) and TLR-2 mediates the induction of proinflammatory response to malaria (32) which is also evident from our results (Fig. 4). The Plasmodium- and Salmonella-infected groups exhibited the induction of expression of TLR-2 and TLR-4. Our results conclude that the higher bacterial load in the coinfected mice is responsible for the overexpression of TLR-4 when compared with only STm-infected mice. Thus, in the condition of coinfection, TLR-2 induced by Pyn along with the over expressed TLR-4 resulted in the overexpression of the downstream Th1/Th2 cytokines (Fig. 5) and NFкB (Fig. 6) ending up in severe inflammatory response induced by STm or Pyn infection.
Th1 cytokines (TNF-α and IFN-γ) represents proinflammatory cytokines stimulating systemic inflammation, whereas Th2 cytokines (IL-4, IL-10, and IL-6) represent anti-inflammatory cytokines that inhibit inflammation and enhance healing (33). Inflammatory responses are essential to control the proliferation of STm but hemolysis caused by malaria induces the HO-1 (Fig. 6, B and C) which impairs the capability of the host immune system to limit the bacterial burden in coinfected mice. Thus, instead of limiting the proliferation of bacteria, the inflammatory response causes more severe damage to the host tissues and organs. Upon controlling the parasitemia and bacterial load, the expression of TLR-2, TLR-4, NFκB, and Th1 decreased, whereas Th2 (anti-inflammatory cytokine) levels remained elevated which might be because the proinflammatory cytokines dominated during sepsis associated with the progression of the parasitemia and the bacteremia and upon clearance the Th2 cytokines dominated for maintaining the homeostasis.
The study concludes that nontyphoidal and typhoidal Salmonella could lead to a fatal condition in malaria patients by increasing the oxidative stress, inflammation, and liver damage, and should be dealt with appropriate combination of antibiotic and antimalarial.
There are still several unknown critical factors associated with the pathogenesis of Plasmodium–Salmonella coinfection and the standardized mice model could be useful for this purpose. As in vitro studies are not appropriate to predict the prognosis of antimalarial and antibacterial agents along with the adjunct therapies in the coinfected conditions, in vivo, the study offers an insight to clinical management. The present mice model offers the control of both parasitemia and bacteremia after establishment of the initial infection which mimic the initial clinical stage of coinfection.
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