Yasuhiro Tsuda

Influence of systemic inflammatory response syndrome on host resistance against bacterial infections.

Objective:To determine the relationship between systemic inflammatory response syndrome (SIRS) and host innate immunities against bacterial infections. Design:Controlled animal study. Setting:University research laboratory. Subjects:Male BALB/c mice, 8–10 wks of age. Interventions:Morbidity and mortality rates of severe SIRS mice were compared with those of mild SIRS mice after infection with Enterococcus faecalis or methicillin-resistant Staphylococcus aureus (MRSA) or exposure to infectious complications induced by cecal ligation and puncture (CLP). In addition, a function of effector cells related to antibacterial innate immunities for these infections was analyzed in these two groups. Furthermore, SCIDbgMN mice (SCIDbg mice depleted of antibacterial effector cells) were reconstituted with effector cells from mild or severe SIRS mice and exposed to various infections. Measurements and Main Results:Severe SIRS mice were greatly susceptible to E. faecalis, MRSA, and CLP-induced sepsis. On the other hand, as compared with normal mice, mild SIRS mice were resistant to these infections. All of SCIDbgMN mice inoculated with peritoneal macrophages (PM&phgr;) from severe SIRS mice died after infection with E. faecalis or MRSA, whereas all SCIDbgMN mice inoculated with PM&phgr; from mild SIRS mice survived after the same infection. SCIDbgMN mice that were inoculated with PM&phgr; from normal mice and exposed to E. faecalis, MRSA, or CLP survived at rates of 50%, 50%, or 60%, respectively. PM&phgr; from mild SIRS mice exhibited typical properties for classically activated macrophages (CAM&phgr;), whereas those from severe SIRS mice exhibited typical properties for alternatively activated macrophages (AAM&phgr;). Conclusions:M&phgr;-associated host antibacterial innate immunities are greatly influenced by SIRS levels. CAM&phgr;, effector cells for the antibacterial innate immunity against E. faecalis, MRSA, and CLP-induced sepsis, are induced in mild SIRS mice. AAM&phgr; with no antibacterial capabilities are generated in mice with severe SIRS. Induction of CAM&phgr; may protect severe SIRS patients against infections.

Bacterial sepsis and chemokines.

Bacterial sepsis causes a high mortality rate when it occurs in patients with compromised host defenses. Severely burned patients, typical immunocompromised hosts, are extremely susceptible to infections from various pathogens, and a local wound infection frequently escalates into sepsis. In these patients, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa are familiar pathogens that cause opportunistic infections. Also, polymicrobial sepsis frequently occurs in these patients. In this review, therefore, the roles of chemokines in thermally injured patients infected with these 3 pathogens and polymicrobial sepsis will be discussed. These infections in thermally injured patients may be controlled immunologically, because immunocompetent hosts are resistant to infections with these pathogens. Classically activated macrophages (M1Mphi) are major effector cells for host innate immune responses against these infections. However, M1Mphi are not generated in thermally injured patients whose alternatively activated macrophages (M2Mphi) predominate. M2Mphi appear in patients early after severe burn injuries. M2Mphi inhibit M1Mphi generation through the secretion of CCL17 and IL-10. As a modulator of Mphi, two different subsets of neutrophils (PMN-I, PMN-II) are described. PMN-I direct the polarization of resident Mphi into M1Mphi through the production of CCL3. M2Mphi are induced from resident Mphi by CCL2 released from PMN-II. Therefore, as an inhibitor of CCL2, glycyrrhizin protects individuals infected with S. aureus. Sepsis stemming from P. aeruginosa wound infection is also influenced by CCL2 released from immature myeloid cells. A large number of immature myeloid cells appear in association with burn injuries. Host resistance to S. aureus, E. faecalis, P. aeruginosa or polymicrobial infections may be improved in thermally injured patients through the induction of M1Mphi, elimination of CCL2 and/or depletion of M2Mphi induced by CCL2.

Role of Polymorphonuclear Neutrophils on Infectious Complications Stemming from Enterococcus faecalis Oral Infection in Thermally Injured Mice

Thermally injured mice are susceptible to Enterococcus faecalis translocation. In this study, the role of polymorphonuclear neutrophils (PMN) on the development of sepsis stemming from E. faecalis translocation was studied in SCID-beige (SCIDbg) mice depleted of PMN (SCIDbgN mice) or macrophages (Mφ) and PMN (SCIDbgMN mice). Sepsis was not developed in SCIDbgN mice orally infected with E. faecalis, while the orally infected pathogen spread systemically in the same mice inoculated with PMN from thermally injured mice (TI-PMN). SCIDbgMN mice were shown to be greatly susceptible to sepsis caused by E. faecalis translocation, while orally infected E. faecalis did not spread into sepsis in the same mice that were previously inoculated with Mφ from unburned SCIDbg mice (resident Mφ). In contrast, orally infected E. faecalis spread systemically in SCIDbgMN mice inoculated with resident Mφ and TI-PMN, while all SCIDbgMN mice inoculated in combination with resident Mφ and PMN from unburned SCIDbg mice survived after the infection. After cultivation with TI-PMN in a dual-chamber transwell, resident Mφ converted to alternatively activated Mφ, which are inhibitory on the generation of classically activated Mφ (typical effector cells in host antibacterial innate immunities). TI-PMN were characterized as immunosuppressive PMN (PMN-II) with abilities to produce cc-chemokine ligand-2 and IL-10. These results indicate that PMN-II appearing in response to burn injury impair host antibacterial resistance against sepsis stemming from E. faecalis translocation through the conversion of resident Mφ to alternatively activated Mφ.

An immunosuppressive subtype of neutrophils identified in patients with hepatocellular carcinoma

Functional disorders of various immune cells have been reported in hepatocellular carcinoma (HCC) patients. Recently, distinct subsets of neutrophils (polymorphonuclear leukocytes, PMN) have been identified in hosts with enhanced or impaired cell-mediated immunity. In this study, therefore, plasma factors and PMN from HCC patients were immunobiologically investigated. Plasma neopterin and CCL17 levels were measured by ELISA in 95 HCC patients. Peripheral PMN were isolated from each HCC patient and tested for CCL2 or CCL3 production by ELISA and flow cytometry. The results showed elevated plasma neopterin levels in HCC patients, while CCL17 levels decreased in correlation with tumor size. PMN from HCC patients produced CCL2, while PMN from healthy subjects did not. Moreover, CCL2 production by PMN was significantly increased in proportion to tumor load. When HCC patients were divided into two groups based on CCL2 produced by PMN, the survival rate of the CCL2 high group was significantly lower than that for other patients. While CCL3 production by PMN was also significantly increased in HCC patients, their CCL3 production did not correlate with tumor load and survival. The CCL2/CCL3 ratio in culture fluids of each PMN was also increased in proportion to tumor size. These results suggest that cell-mediated immunity may be impaired in advanced HCC patients. Moreover, distinct PMN subsets may exist in the peripheral blood of HCC patients. These PMN subsets, especially CCL2-producing PMN, may be involved in tumor extension and the survival outcomes for HCC patients.

Evaluation of Portal Hypertensive Enteropathy by Scoring with Capsule Endoscopy: Is Transient Elastography of Clinical Impact?

There is limited data about the mucosal lesions of portal hypertensive enteropathy (PHE) detected by capsule endoscopy, and there is no scoring system to evaluate their severity. Our aim is to create a reliable scoring system for PHE, and to explore the possible usefulness of using transient elastograhy (TE) in that field. We compared the medical records of 31 patients with liver cirrhosis and portal hypertension with 29 control patients. We found that the mucosal lesions compatible with PHE were significantly more common in cirrhotic patients than in control patients (67.7% vs 6.9%, p<0.001). Cirrhotic patients with high TE score (p = 0.018), high Child-Pugh grade, large esophageal varices (EV), portal hypertensive gastropathy, and history of endoscopic variceal injection sclerotherapy or ligation (EIS/EVL) were significantly associated with PHE. Using our scoring system, we found that patients with higher TE score (p = 0.004), high Child-Pugh score (p = 0.011), larger EV (p = 0.006), and prior EIS/EVL (p = 0.006) were significantly associated with higher PHE score. We concluded that using our scoring system might be helpful in grading PHE severity, and TE might be a new non-invasive method for detecting the presence and severity of PHE in cirrhotic patients.

CCL2, a product of mice early after systemic inflammatory response syndrome (SIRS), induces alternatively activated macrophages capable of impairing antibacterial resistance of SIRS mice

Infection associated with systemic inflammatory response syndrome (SIRS) is a major cause of morbidity and mortality in patients with major surgery, polytrauma, and severe burn injury. In previous studies, mice with severe pancreatitis (a mouse model of SIRS, SIRS mice) have been shown to be greatly susceptible to various infections. In the present study, a mechanism involved in the impaired resistance of SIRS mice to infectious complications was investigated. Sera from SIRS mice impaired the resistance of normal mice to infectious complications induced by cecal ligation and puncture (CLP). CC chemokine ligand 2 (CCL2) was detected in sera of SIRS mice. Resident macrophages (RMφ) cultured with SIRS mouse sera converted to alternatively activated macrophages (AAMφ), which were also demonstrated in mice treated with recombinant murine CCL2. However, AAMφ were not demonstrated in mice injected with SIRS mouse sera and anti‐CCL2 monoclonal antibody (mAb) in combination. Furthermore, normal mice that received SIRS mouse sera and anti‐CCL2 mAb resisted CLP‐induced infectious complications. These results indicate that the resistance of SIRS mice to infectious complications is impaired by AAMφ generated from RMφ in response to SIRS‐associated CCL2 production.

Lipopolysaccharides accelerate hepatic steatosis in the development of nonalcoholic fatty liver disease in Zucker rats

Nonalcoholic fatty liver disease (NAFLD) can develop into end-stage disease that includes cryptogenic cirrhosis and hepatocellular carcinoma. Bacterial endotoxin, for example lipopolysaccharide (LPS), plays an important role in the pathogenesis of NAFLD. The aim of this study was to assess the role of LPS in the development of NAFLD. Twenty-one male Zucker (fa/fa) rats were divided into three groups: rats fed for twelve weeks on a diet rich in disaccharide (D12 group), rats similarly managed but treated with LPS (LPS group), and those on the same diet for 24 weeks (D24 group). Histological examination demonstrated that this protocol induced hepatic steatosis in the LPS and D24 groups. Significant, marked accumulation of lipid droplets was observed in the LPS group, compared with the D24 group. Rats from the LPS group showed a decrease in plasma adiponectin levels, an increase in plasma leptin levels, and greater expression of FAS and SREBP-1c mRNA in the liver, compared with rats from the D24 group. These finding coincided with histological findings. We therefore suggest that LPS may accelerate the progression of hepatic steatosis.

CCL2 as a trigger of manifestations of compensatory anti-inflammatory response syndrome in mice with severe systemic inflammatory response syndrome

Patients with compensatory anti‐inflammatory response syndrome (CARS) are at a higher risk for infection with various opportunistic pathogens. CARS develops commonly in association with the manifestation of systemic inflammatory response syndrome (SIRS). In the present study, the role of SIRS‐associated soluble factors on the CARS development was examined in mice with pancreatitis, a carrier of typical SIRS. Following the production of SIRS‐related cytokines [tumor necrosis factor α and interleukin (IL)‐1β], CC chemokine ligand 2 (CCL2), IL‐4, and IL‐10 (typical CARS cytokines) were detected in the sera of mice with pancreatitis. CCL2 has been described as an essential chemokine for the T helper cell type 2 manifestation. CARS effector cells (cells with an ability to produce IL‐4 and IL‐10) were not generated from normal T cells after stimulation with SIRS‐related cytokines. However, these cells were generated from normal T cells after cultivation with peripheral blood neutrophils (PMN) from SIRS mice in a dual‐chamber transwell. Normal T cells did not convert to CARS effector cells after transwell cultures with PMN from normal mice. CCL2 was detected in culture fluids of PMN from SIRS mice, and PMN from normal mice did not produce CCL2 into their culture fluids. CARS effector cells did not appear in PMN‐depleted SIRS mice or SIRS mice treated with anti‐CCL2 monoclonal antibody, and these cells were demonstrated in PMN‐depleted SIRS mice after treatment with recombinant murine CCL2. These results indicate that CCL2 produced by PMN from SIRS mice is an active molecule on the SIRS‐associated CARS manifestation.

Glycyrrhizin inhibits neutrophil-associated generation of alternatively activated macrophages

Abstract Patients with severe burn injuries are extremely susceptible to infection, and the hosts antibacterial responses are frequently suppressed by alternatively activated macrophages (M2Mϕ), commonly demonstrated in these patients. An immunosuppressive subset of neutrophils (PMN-II), demonstrated in the peripheral blood of thermally injured patients, has been described as an inducer of M2Mϕ. In the present studies, the inhibitory effect of glycyrrhizin (GL) on M2Mϕ generation stimulated by PMN-II was examined. M2Mϕ were generated from resident Mϕ (R-Mϕ, lower chamber) after cultivation with PMN-II (upper chamber) in a dual-chamber transwell. However, M2Mϕ were not generated from R-Mϕ when the same transwell cultures were performed in the presence of GL. M2Mϕ were not generated from R-Mϕ after cultivation with PMN-II previously treated with GL, while R-Mϕ previously treated with GL converted to M2Mϕ after they were cultured with PMN-II in transwells. Interleukin-10 and CCL2 released from PMN-II were shown to be effector molecules responsible for the generation of M2Mϕ. However, these soluble factors were not produced by PMN-II treated with GL. These results indicate that GL inhibits PMN-II-stimulated M2Mϕ generation through the inhibition of CCL2/interleukin-10 production by PMN-II.

Pathogenic Role of Macrophages in Intradermal Infection of Methicillin-Resistant Staphylococcus aureus in Thermally Injured Mice

ABSTRACT Intradermal infection of methicillin-resistant Staphylococcus aureus (MRSA) in burned mice was pathogenically analyzed. An abscess was formed in normal mice intradermally infected with 108 CFU/mouse of MRSA, and all of these mice survived after the infection; however, abscess formation was not demonstrated to occur in burned mice similarly exposed to the pathogen, and all of these mice died within 5 days of infection. In burned mice, MRSA infected at the burn site intradermal tissues spread quickly throughout the whole body, while in normal mice, the pathogen remained localized at the infection site. Macrophages (Mφ) isolated from the infection site tissues of normal mice produced interleukin-12 (IL-12) but not IL-10 and were characterized as M1Mφ. These M1Mφ were not isolated from the infection site tissues of burned mice. When normal-mouse infection site tissue Mφ were adoptively transferred to burned mice at the MRSA infection site, an abscess formed, and the infection did not develop into sepsis. In contrast, an abscess did not form and sepsis developed in normal mice that were inoculated with burned-mouse infection site tissue Mφ. These Mφ produced IL-10 but not IL-12 and were characterized as M2Mφ. These results indicate that abscess formation is a major mechanism of host resistance against intradermal MRSA infection. M1Mφ in the tissues surrounding the infection site play a pivotal role in abscess formation; however, the abscess is not formed in burned mice where M2Mφ predominate. M2Mφ have been described as inhibitor cells for Mφ conversion from resident Mφ to M1Mφ.