Orthopaedic Surgery Elicits a Systemic Anti-Inflammatory Signature.

Little information is available on the functional activity of leukocytes after arthroplasty or the expansion of populations with immune suppressive properties during the acute post-operative period. Synovial fluid and matched pre- and post-surgical blood samples were collected from total hip and knee arthroplasty patients (THA and TKA, respectively) to examine the impact of surgery on peripheral blood leukocyte frequency, bactericidal activity, and inflammatory mediator expression. For spinal surgeries, inflammatory mediator production by peripheral blood mononuclear cells (PBMCs) pre- and post-surgery was examined. An expansion of immune suppressive granulocytic myeloid-derived suppressor cells (G-MDSCs) was observed following arthroplasty, which correlated with significantly increased serum interleukin-10 (IL-10) levels. Analysis of synovial fluid from THA and TKAs revealed reduced granulocyte colony-stimulating factor (G-CSF) and soluble CD40 ligand (sCD40L) and increased interleukin-6 (IL-6), monocyte chemoattractant protein 2 (CCL2) and Fms-like tyrosine kinase 3 ligand (Flt-3L) compared to pre- and post-surgical serum. For the spinal surgery cohort, stimulation of PBMCs isolated post-surgery with bacterial antigens produced significantly less pro-inflammatory (IL-1α, IL-1ß, interleukin-1 receptor antagonist (IL-1RA), IL-12p40, growth-related oncogene-α/GRO-α (CXCL1) and 6Ckine (CCL21)) and more anti-inflammatory/tissue repair mediators (IL-10, G-CSF and granulocyte-macrophage colony-stimulating factor (GM-CSF)) compared to PBMCs recovered before surgery. The observed bias towards systemic anti-inflammatory changes without concomitant increases in pro-inflammatory responses may influence susceptibility to infection following orthopaedic surgery in the context of underlying co-morbidities or risk factors.

Human prosthetic joint infections are associated with myeloid-derived suppressor cells (MDSCs): Implications for infection persistence.

Prosthetic joint infection (PJI) is a devastating complication of joint arthroplasty surgery typified by biofilm formation. Currently, mechanisms whereby biofilms persist and evade immune-mediated clearance in immune competent patients remain largely ill-defined. Therefore, the current study characterized leukocyte infiltrates and inflammatory mediator expression in tissues from patients with PJI compared to aseptic loosening. CD33+ HLA-DR- CD66b+ CD14-/low granulocytic myeloid-derived suppressor cells (G-MDSCs) were the predominant leukocyte population at sites of human PJI compared to aseptic tissues. MDSCs inhibit T cell proliferation, which coincided with reduced T cells in PJIs compared to aseptic tissues. IL-10, IL-6, and CXCL1 were significantly elevated in PJI tissues and have been implicated in MDSC inhibitory activity, expansion, and recruitment, respectively, which may account for their preferential increase in PJIs. This bias towards G-MDSC accumulation during human PJI could account for the chronicity of these infections by preventing the pro-inflammatory, antimicrobial actions of immune effector cells. CLINICAL SIGNIFICANCE: Animal models of PJI have revealed a critical role for MDSCs and IL-10 in promoting infection persistence; however, whether this population is prevalent during human PJI and across distinct bacterial pathogens remains unknown. This study has identified that granulocytic-MDSC infiltrates are unique to human PJIs caused by distinct bacteria, which are not associated with aseptic loosening of prosthetic joints. Better defining the immune status of human PJIs could lead to novel immune-mediated approaches to facilitate PJI clearance in combination with conventional antibiotics. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1605-1613, 2018.

Infectious Dose Dictates the Host Response during Staphylococcus aureus Orthopedic-Implant Biofilm Infection.

Staphylococcus aureus is a leading cause of prosthetic joint infections (PJIs) that are typified by biofilm formation. Given the diversity of S. aureus strains and their propensity to cause community- or hospital-acquired infections, we investigated whether the immune response and biofilm growth during PJI were conserved among distinct S. aureus clinical isolates. Three S. aureus strains representing USA200 (UAMS-1), USA300 (LAC), and USA400 (MW2) lineages were equally effective at biofilm formation in a mouse model of PJI and elicited similar leukocyte infiltrates and cytokine/chemokine profiles. Another factor that may influence the course of PJI is infectious dose. In particular, higher bacterial inocula could accelerate biofilm formation and alter the immune response, making it difficult to discern underlying pathophysiological mechanisms. To address this issue, we compared the effects of two bacterial doses (10(3) or 10(5) CFU) on inflammatory responses in interleukin-12p40 (IL-12p40) knockout mice that were previously shown to have reduced myeloid-derived suppressor cell recruitment concomitant with bacterial clearance after low-dose challenge (10(3) CFU). Increasing the infectious dose of LAC to 10(5) CFU negated these differences in IL-12p40 knockout animals, demonstrating the importance of bacterial inoculum on infection outcome. Collectively, these observations highlight the importance of considering infectious dose when assessing immune responsiveness, whereas biofilm formation during PJI is conserved among clinical isolates commonly used in mouse S. aureus infection models.

Interleukin-10 production by myeloid-derived suppressor cells contributes to bacterial persistence during Staphylococcus aureus orthopedic biofilm infection.

Staphylococcus aureus is known to establish biofilms on medical devices. We recently demonstrated that Ly6G(high)Ly6C(+) myeloid-derived suppressor cells are critical for allowing S. aureus biofilms to subvert immune-mediated clearance; however, the mechanisms whereby myeloid-derived suppressor cells promote biofilm persistence remain unknown. Interleukin-10 expression was significantly increased in a mouse model of S. aureus orthopedic implant biofilm infection with kinetics that mirrored myeloid-derived suppressor cell recruitment. Because myeloid-derived suppressor cells produce interleukin-10, we explored whether it was involved in orchestrating the nonproductive immune response that facilitates biofilm formation. Analysis of interleukin-10-green fluorescent protein reporter mice revealed that Ly6G(high)Ly6C(+) myeloid-derived suppressor cells were the main source of interleukin-10 during the first 2 wk of biofilm infection, whereas monocytes had negligible interleukin-10 expression until day 14. Myeloid-derived suppressor cell influx into implant-associated tissues was significantly reduced in interleukin-10 knockout mice at day 14 postinfection, concomitant with increased monocyte and macrophage infiltrates that displayed enhanced proinflammatory gene expression. Reduced myeloid-derived suppressor cell recruitment facilitated bacterial clearance, as revealed by significant decreases in S. aureus burdens in the knee joint, surrounding soft tissue, and femur of interleukin-10 knockout mice. Adoptive transfer of interleukin-10 wild-type myeloid-derived suppressor cells into S. aureus-infected interleukin-10 knockout mice restored the local biofilm-permissive environment, as evidenced by increased bacterial burdens and inhibition of monocyte proinflammatory activity. These effects were both interleukin-10-dependent and interleukin-10-independent because myeloid-derived suppressor cell-derived interleukin-10 was required for promoting biofilm growth and anti-inflammatory gene expression in monocytes but was not involved in monocyte recruitment to biofilm-infected tissues. These results demonstrate that interleukin-10 production by myeloid-derived suppressor cells contributes to the persistence of S. aureus orthopedic biofilm infections.

IL-12 promotes myeloid-derived suppressor cell recruitment and bacterial persistence during Staphylococcus aureus orthopedic implant infection.

Staphylococcus aureus is a leading cause of human prosthetic joint infections (PJIs) typified by biofilm formation. We recently identified a critical role for myeloid-derived suppressor cells (MDSCs) in S. aureus biofilm persistence. Proinflammatory signals induce MDSC recruitment and activation in tumor models; however, the mechanisms responsible for MDSC homing to sites of biofilm infection are unknown. In this study, we report that several cytokines (IL-12p40, IL-1ß, TNF-α, and G-CSF) and chemokines (CXCL2, CCL5) were significantly elevated in a mouse model of S. aureus PJI. This coincided with significantly increased MDSC infiltrates concomitant with reduced monocyte, macrophage, and T cell influx compared with uninfected animals. Of the cytokines detected, IL-12 was of particular interest based on its ability to possess either pro- or anti-inflammatory effects mediated through p35-p40 heterodimers or p40 homodimers, respectively. MDSC recruitment was significantly reduced in both p40 and p35 knockout mice, which resulted in enhanced monocyte and neutrophil influx and bacterial clearance. Adoptive transfer of wild-type MDSCs into infected p40 knockout animals worsened disease outcome, as evidenced by the return of S. aureus burdens to levels typical of wild-type mice. Tissues obtained from patients undergoing revision surgery for PJI revealed similar patterns of immune cell influx, with increased MDSC-like cells and significantly fewer T cells compared with aseptic revisions. These findings reveal a critical role for IL-12 in shaping the anti-inflammatory biofilm milieu by promoting MDSC recruitment.

Myeloid-derived suppressor cells contribute to Staphylococcus aureus orthopedic biofilm infection.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature monocytes and granulocytes that are potent inhibitors of T cell activation. A role for MDSCs in bacterial infections has only recently emerged, and nothing is known about MDSC function in the context of Staphylococcus aureus infection. Because S. aureus biofilms are capable of subverting immune-mediated clearance, we examined whether MDSCs could play a role in this process. CD11b(+)Gr-1(+) MDSCs represented the main cellular infiltrate during S. aureus orthopedic biofilm infection, accounting for >75% of the CD45+ population. Biofilm-associated MDSCs inhibited T cell proliferation and cytokine production, which correlated with a paucity of T cell infiltrates at the infection site. Analysis of FACS-purified MDSCs recovered from S. aureus biofilms revealed increased arginase-1, inducible NO synthase, and IL-10 expression, key mediators of MDSC suppressive activity. Targeted depletion of MDSCs and neutrophils using the mAb 1A8 (anti-Ly6G) improved bacterial clearance by enhancing the intrinsic proinflammatory attributes of infiltrating monocytes and macrophages. Furthermore, the ability of monocytes/macrophages to promote biofilm clearance in the absence of MDSC action was revealed with RB6-C85 (anti-Gr-1 or anti-Ly6G/Ly6C) administration, which resulted in significantly increased S. aureus burdens both locally and in the periphery, because effector Ly 6C monocytes and, by extension, mature macrophages were also depleted. Collectively, these results demonstrate that MDSCs are key contributors to the chronicity of S. aureus biofilm infection, as their immunosuppressive function prevents monocyte/macrophage proinflammatory activity, which facilitates biofilm persistence.

Differential effects of interleukin-17 receptor signaling on innate and adaptive immunity during central nervous system bacterial infection.

Although IL-17A (commonly referred to as IL-17) has been implicated in the pathogenesis of central nervous system (CNS) autoimmune disease, its role during CNS bacterial infections remains unclear. To evaluate the broader impact of IL-17 family members in the context of CNS infection, we utilized IL-17 receptor (IL-17R) knockout (KO) mice that lack the ability to respond to IL-17, IL-17F and IL-17E (IL-25). In this article, we demonstrate that IL-17R signaling regulates bacterial clearance as well as natural killer T (NKT) cell and gamma-delta (γδ) T cell infiltrates during Staphylococcus aureus-induced brain abscess formation. Specifically, when compared with wild-type (WT) animals, IL-17R KO mice exhibited elevated bacterial burdens at days 7 and 14 following S. aureus infection. Additionally, IL-17R KO animals displayed elevated neutrophil chemokine production, revealing the ability to compensate for the lack of IL-17R activity. Despite these differences, innate immune cell recruitment into brain abscesses was similar in IL-17R KO and WT mice, whereas IL-17R signaling exerted a greater influence on adaptive immune cell recruitment. In particular, γδ T cell influx was increased in IL-17R KO mice at day 7 post-infection. In addition, NK1.1high infiltrates were absent in brain abscesses of IL-17R KO animals and, surprisingly, were rarely detected in the livers of uninfected IL-17R KO mice. Although IL-17 is a key regulator of neutrophils in other infection models, our data implicate an important role for IL-17R signaling in regulating adaptive immunity during CNS bacterial infection.

Roles of Toll-like receptor 2 (TLR2) and superantigens on adaptive immune responses during CNS staphylococcal infection.

Staphylococcus aureus is a common etiologic agent of brain abscesses and possesses numerous virulence factors that manipulate host immunity. One example is superantigens (SAG) that clonally expand T cell subsets bearing specific Vß receptors. Toll-like receptor 2 (TLR2) is one receptor implicated in S. aureus recognition. However, the interplay between TLR2, SAG, and adaptive immunity during brain abscess formation has not yet been investigated and could reveal novel insights into host-pathogen interactions for regulating protective immunity. A comprehensive analysis of abscess-associated T cell populations in TLR2 KO and WT mice was performed following infection with a S. aureus clinical isolate. Both natural killer T (NKT) and γδ T cell infiltrates were increased in brain abscesses of TLR2 KO mice and produced more IL-17 and IFN-γ compared to WT populations, which could have resulted from elevated bacterial burdens observed in these animals. Analysis of SAG-reactive T cells revealed a predominant Vß(8.1,8.2) infiltrate reactive with staphylococcal enterotoxin B (SEB), whereas SEA-reactive Vß(11) T cells were less numerous. Brain abscesses of TLR2 KO mice had fewer Vß(8.1,8.2) and Vß(11) T cells and produced less TNF-α and IFN-γ compared to WT animals. Treatment of primary microglia with purified SEB augmented TNF-α production in response to the TLR2 ligand Pam3Cys, which may serve to amplify proinflammatory cascades during CNS S. aureus infection. Collectively, these studies demonstrate that TLR2 impacts adaptive immunity to S. aureus infection and modulates SAG responses.

TLR2 deficiency leads to increased Th17 infiltrates in experimental brain abscesses.

TLR2 plays a pivotal role in recognizing Staphylococcus aureus, a common etiologic agent of CNS parenchymal infections, such as brain abscess. We previously reported that brain abscesses of TLR2 knockout (KO) mice exhibited elevated IL-17 levels, suggesting the presence of an alternative pathway available to respond to S. aureus infection that may involve Th17 cells. Both CD4(+) and CD8(+) T cell infiltrates were elevated in brain abscesses of TLR2 KO mice at days 3, 7, and 14 postinfection compared with wild-type animals. Intracellular cytokine staining revealed a significant increase in the frequency of IL-17-producing Th17 cells in TLR2 KO mice with relatively few IFN-gamma-positive cells. gammadelta T cells were also a source of IL-17 in brain abscesses. Microglia, astrocytes, and macrophages were shown to express both IL-17RA and IL-17RC. Despite receptor expression, IL-17 was relatively ineffective at eliciting glial activation, whereas the cytokine augmented the ability of TNF-alpha to induce CXCL2 and CCL2 expression by macrophages. Based on the ability of IL-17 to elicit the release of chemokines and other proinflammatory mediators, we propose that the exaggerated IL-17 response that occurs in TLR2 KO mice functions in a compensatory manner to control brain abscess pathogenesis, with cells other than glia as targets for IL-17 action. This is supported by our findings in which innate immune infiltrates were not significantly different between TLR2 KO and wild-type mice in conjunction with the lack of prolonged alterations in the synthesis of other proinflammatory molecules during the course of infection.

Association of chronic alcohol consumption and increased susceptibility to and pathogenic effects of pulmonary infection with respiratory syncytial virus in mice.

Chronic alcohol abuse by human beings has been shown to be associated with increased susceptibility to pulmonary infections and severity of inflammatory responses associated with pulmonary infection. On the basis of the higher likelihood of exposure to respiratory viruses, people who abuse alcohol would logically be susceptible to respiratory viral infections. To test this hypothesis, mice were provided alcohol in drinking water for 13-16 weeks with the Meadows-Cook protocol and infected intranasally with respiratory syncytial virus. At various times after infection, severity of infection was determined by evaluation of cellular and cytokine composition of bronchoalveolar lavage fluid (BALF) and histologic evaluation of inflammation. Infection was associated with neutrophil infiltration in both groups, but the proportion and number of neutrophils in BALF were significantly greater in the alcohol consumption group than in the control group. Concentrations of tumor necrosis factor-alpha and monocyte chemoattractant protein-1 in BALF in the alcohol consumption group were increased. Interferon (IFN)-gamma concentrations were lower in the alcohol consumption group at later times of infection. Pulmonary inflammation was cleared by 3-5 days after infection in the control group. In contrast, pulmonary inflammation was evident in the alcohol consumption group after 7 days of infection, and some mice showed severe inflammation with hemorrhage and edema. IFN-alpha/beta was evident in BALF at low concentrations in the alcohol consumption group for several days after infection, and increased mRNA for IFN-alpha/beta was also evident in the alcohol consumption group. This was accompanied by the presence of virus in this group at these times of infection. Chronic alcohol consumption increased severity of pulmonary infection with a virus that naturally infects hosts by an aerosol route. Infection of mice that had consumed alcohol chronically was more severe in terms of increased proinflammatory cytokine production, inflammation, and a failure to clear the virus from the lungs.

Alcoholic pancreatitis: mechanisms of viral infections as cofactors in the development of acute and chronic pancreatitis and fibrosis.

Acute and chronic pancreatitis is associated with alcohol abuse, but symptomatic pancreatitis develops in only a small proportion of persons (10-20%) who abuse alcohol. This apparent paradox has led to the notion that additional cofactors are involved in the development of alcoholic pancreatitis. Potential cofactors, such as diet and smoking, have been suggested, but there are no compelling epidemiologic data to support this idea. A number of viruses and some bacteria have been shown to infect the pancreas and produce pancreatitis. One important mediator of pancreatitis in persons with a compromised immune system is a viral infection. The increased susceptibility of immunocompromised persons to viral pancreatitis led to the hypothesis, described in this paper, that the well-known immunosuppression associated with alcohol abuse would result in a more severe viral pancreatitis in mice, which are provided ethanol, than in control animals. To test this hypothesis, C57BL/6 mice were infected with a virulent strain of coxsackievirus B3, which preferentially induces pancreatitis, or with a strain that is naturally avirulent. The study findings presented in this paper show that ethanol consumption alone does not produce pancreas damage but results in a more severe and prolonged pancreatitis after infection with a virulent virus and interestingly, after infection with the avirulent strain of virus. This was associated with an increased number of viruses in the pancreas and spleen, which correlated with decreased humoral immune responses to the virus.

Rescue of in vivo FAS-induced apoptosis of hepatocytes by corticosteroids either associated with alcohol consumption by mice or provided exogenously.

The pathogenic effects of many hepatic viral infections are mediated, at least in part, by the immune response to the infected hepatocyte. The immune response in the infected liver involves the interaction of cytotoxic T cells (CTL) with the hepatocytes through the interaction of FAS-ligand on the CTL and FAS on the hepatocyte. The initial hypothesis for this study was that alcohol consumption by mice would sensitize the liver to apoptosis induced by ligation of FAS. C57Bl/6 mice fed ethanol in a liquid diet did show an increased percentage of apoptotic cells 2 h after injection with anti-FAS as compared with the percentage in the control mice. However, 4 and 6 h after anti-FAS injection, control mice showed high percentages of apoptotic cells (20% to 41%) compared with 5% and 4% apoptotic cells in the ethanol-fed mice. The decreased apoptosis of ethanol-fed mice correlated closely with corticosterone levels in the sera. This was confirmed by the finding that adrenalectomized (ADX) mice provided a high level of corticosterone in drinking water were protected against FAS-induced hepatocyte apoptosis. Ethanol-fed mice showed a significant elevation of serum alanine aminotransferase (ALT) levels indicating the development of hepatitis in spite of the relatively low proportion of apoptotic cells in the liver. In conclusion, high levels of corticosterone protect hepatocytes from FAS-mediated apoptosis, but do not prevent the ultimate development of liver damage. In experiments where mice were provided ethanol chronically in drinking water, where stress is minimal, higher levels of ALT were noted in animals in the ethanol group as compared with animals in the control group. These data support the suggestion that ethanol increases hepatocyte sensitivity to FAS-mediated damage.