Innate immune recognition of, and response to, Clostridium sordellii

Anaerobe 16 (2010) 125–130

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Microbial/host interactions

Innate immune recognition of, and response to, Clostridium sordellii M.J. Aldape a, *, A.E. Bryant a, E.J. Katahira a, A.M. Hajjar b, S.M. Finegold c, Y. Ma a, d, D.L. Stevens a, b a

Veterans Affairs Medical Center, Infectious Diseases Section, 500 West Fort St. (Bldg 45), Boise, ID 83702, USA University of Washington, Seattle, WA, USA c Veterans Affairs Medical Center, Los Angeles, CA, USA d Idaho State University, Pocatello, ID, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 February 2009 Received in revised form 17 April 2009 Accepted 16 June 2009 Available online 25 June 2009

Clostridium sordellii, an anaerobic pathogen, has recently been associated with rapidly fatal infections following medically induced abortions and injecting drug use. Patients with C. sordellii infection display few signs of inflammation such as fever, or redness and pain at the site of infection. We hypothesized that this could be due to reduced recognition of the organism by Toll-like receptors (TLRs) of the innate immune system. An ELAM-NF-kB luciferase reporter system in TLR-transfected HEK cells was used to measure TLR-dependent recognition of washed, heat-killed C. sordellii and other pathogenic clostridial species. Results demonstrated that all clostridia were well recognized by TLR2 alone and that responses were greatest when TLR2 was co-expressed with TLR6. Further, isolated human monocytes produced the pro-inflammatory cytokine TNFa and the immunoregulator IL-10 in response to C. sordellii. In addition, C. sordellii-stimulated monocytes produced 30% less TNFa following treatment with an anti-TLR2 blocking antibody. These data demonstrate that innate immune recognition of, and response to, cellassociated components of C. sordellii and other clostridial pathogens are mediated by TLR2 in combination with TLR6. We conclude that the characteristic absence of inflammatory signs and symptoms in C. sordellii infection is not related to inadequate immune detection of the organism, but rather is attributable to a species-specific immune system dysfunction that remains to be elucidated. Published by Elsevier Ltd.

Keywords: Clostridium sordellii Toll-like receptors Innate immune system

1. Introduction Clostridium sordellii is an anaerobic Gram-positive bacillus that causes aggressive necrotizing infections in previously healthy humans, most of which are fatal (70%) [1]. C. sordellii is ubiquitously distributed in nature and can be isolated from the soil and gastrointestinal flora of several animals including humans. Infections are most commonly associated with childbirth and gynecological procedures but may also develop following traumatic injury or surgery [1]. Recently, life-threatening C. sordellii infections have been described in injecting-drug users [2,3] and in women undergoing medically-induced abortions with mifepristone/misoprostol [4–6]. In patients with C. sordellii infection, fever is typically absent and only mild pain and limited inflammation is observed at sites of infection [1]. The absence of a robust inflammatory response often confuses physicians and results in misdiagnosis, delays in treatment and ultimately increased mortality. The underlying mechanisms responsible for this poor inflammatory host response remain entirely unknown.

* Corresponding author. Tel.: þ1 208 422 1599; fax: þ1 208 422 1365. E-mail address: [email protected] (M.J. Aldape). 1075-9964/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.anaerobe.2009.06.004

The innate immune system represents the first line of host defense against invading microbes and controls the initiation of the adaptive immune response. The family of Toll-like receptors (TLRs) has proven to be essential to the innate immune response and plays critical roles in both detecting and signaling infection (reviewed in [7,8]). TLRs are evolutionary conserved molecules that are recognized as type I transmembrane proteins. They contain an extracellular leucine-rich repeat domain and an intracellular C-terminal signaling domain with sequence homology to the human interleukin-1 (IL-1) receptor. As the main sensors of the innate immune system, engagement of TLRs on monocytes/macrophages and other antigen presenting cells triggers signaling cascades resulting in the activation of the transcription factor NF-kB. NF-kB is a pivotal regulator of the innate immune response that controls production of several pro-inflammatory cytokines (e.g., TNFa, IL-1b, IL-6) important in host defense [9]. TLRs recognize highly conserved structures of bacterial and viral origin known as pathogen-associated molecular patterns (PAMPs) [7]. While not completely understood, many of the ligands for the different TLRs have been determined. For example, TLR2 recognizes components of Gram-positive organisms including lipoteichoic acid (LTA) and peptidoglycan [10]. TLR4 mediates responses to lipopolysaccharide (LPS) from Gram-negative organisms and TLR3

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recognizes double-stranded RNA released during viral infection [7]. TLR5 binds to flagellin, a protein monomer of the flagellum of motile bacteria [11]. The direct involvement of TLRs in the hosts innate immune response to C. sordellii (or any clostridial species) has never been reported. Therefore, this study (1) investigated which TLRs were utilized for innate immune recognition of C. sordellii, (2) determined the magnitude of TLR-dependent recognition of C. sordellii, (3) determined whether engagement of C. sordellii by cells associated with the innate immune system resulted in pro-inflammatory cytokine production, and (4) investigated the TLRs utilized for recognition of other pathogenic clostridium species. TLR receptor usage and cytokine production in response to washed, heat killed clostridia were studied using an ELAM-NFkB luciferase reporter system in TLR-transfected HEK cells and in isolated human mononuclear cells, respectively.

(3) selected dilutions of washed, heat killed clostridia; or (4) sterile saline vehicle control. After 4 hrs, the overlying media was removed and saved for IL-8 cytokine analysis by commercial ELISA (R&D Systems, Minneapolis, MN). The HEK293 cells were then lysed and luciferase activity was quantified using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) using a Fluoroskan Ascent fluorometer (Helsinki, Finland). Firefly luciferase units were divided by Renilla luciferase units to obtain relative luciferase units (RLU) and data are reported as the means of triplicate measurements. Data is reported as IL-1b-normalized Relative Luminescence Units [RLU ¼ (ELAM-luciferase/Renilla luciferase) O IL-1b response]. The experiment was performed in triplicate on 3 occasions with the C. perfringens, C. sordellii, and C. septicum and once with C. bolteae and C. clostridioforme.

2.3. Mononuclear cell-derived cytokine production 2. Materials and methods 2.1. Organisms Standard laboratory strains of C. sordellii, Clostridium perfringens, Clostridium septicum and Clostridium clostridioforme were purchased from the American Type Culture Collection (ATCC #9714, #13124, #11424 and #25537 respectively). The Clostridium bolteae strain was a clinical isolate obtained from the stool of a child with autism (WAL# 16351) [12]. A Bactron II anaerobic chamber (Sheldon Manufacturing Inc, Cornelius, OR) was utilized to maintain an anaerobic environment. Organisms were grown to log phase in proteose peptone media as previously described [13], collected by centrifugation and washed and resuspended in endotoxin-free sterile saline. Prepared organisms were heat-killed at 56  C for 60 min, aliquoted and frozen at -70  C until use. Killing was confirmed by plating an undiluted sample of prepared organisms onto Brucella blood agar followed by anaerobic incubation for 24 h. Bacterial preparations were tested for lipopolysaccharide (LPS) contamination by Limulus amebocyte lysate (LAL) assay (Associates of Cape Cod, Woods Hole, MA) and only those with LPS levels < 0.03 EU/mL were utilized. On the day of the experiment, the concentration of stock organisms was adjusted in sterile saline to an optical density at 600 nm of 1.5 (w5  108 organisms/mL) and dilutions thereafter were made in HEK293 cell culture media (see below).

Heparinized whole blood was obtained from healthy human volunteers who gave signed informed consent in accordance with the University of Washingtons Human Subjects Division/Institutional Review Board and approved by the Research & Development Service of the Veterans Affairs Medical Center, Boise, ID. Volunteers denied consumption of any prescription or over-the-counter medications in the 10 days prior to the donation. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation as previously described [16] and adjusted to 2  106 cells/mL in RPMI þ 5% pooled human serum. One milliliter of cells was placed into each well of a 24-well tissue culture plate and cells were stimulated for 16 h with either nothing, Salmonella minnesota LPS (10 ng/mL), or washed, heat-killed organisms at OD600nm ¼ 0.014 final concentration. The overlying culture media was collected, centrifuged to remove any detached cells and residual bacteria, and the sample frozen at -70  C until assayed for TNFa and IL-10 by commercial ELISA (R&D Systems). For TLR blocking studies, human PBMCs were prepared as described above and pre-treated for 1 h with either nothing, antihuman TLR2 mAb (20 mg/mL), anti-human TLR4 mAb (20 mg/mL) or an isotype-matched IgG control antibody (20 mg/mL) (all purchased from eBioscience, Inc., San Diego, CA). Following TLR blocking, cells were stimulated with either nothing, LPS or washed, heat-killed C. sordellii (see above). After 16 h, the overlying culture supernatants were collected and assayed for TNFa by commercial ELISA.

2.2. TLR transfectants and ELAM-1 reporter system 3. Results Toll-like receptor utilization assays followed procedures described by Hajjar et al. [14]. Briefly, on the day prior to transfection, HEK293 cells (ATCC, Manassas, VA) were seeded in 96 well plates at a concentration of 4  104 cells/well in DMEM (Gibco, Rockville, MD) with 10% heat inactivated FBS (complete DMEM media; Hyclone, Logan, UT). The ELAM-luciferase reporter plasmid and plasmids encoding for b-actin Renilla luciferase, murine (mu)CD14 and muMD2 were transiently transfected into HEK293 cells using a calcium phosphate precipitation protocol [15] along with muTLR1, muTLR2, muTLR4 and muTLR6, either alone or in combination. Precipitated DNA complexes were added to HEK293 cells and incubated for 4 h. The total amount of DNA transfected per well (2 mg plus the reporter constructs) was normalized by the addition of empty vector. The media was removed, the cells washed once and complete DMEM was added. The following day fresh complete DMEM was added and the cells were stimulated with one of the following: (1) the HEK293 cell agonist, IL-1b (10 ng/mL, Pierce Endogen, Rockford, IL); (2) the TLR4-specific agonist, Salmonella minnesota LPS (10 ng/mL, Sigma, St. Louis, MO);

3.1. C. sordellii TLR usage patterns To investigate the role of TLRs in innate immune recognition of C. sordellii, we examined the clostridia-induced activation of an NF-kB-dependent luciferase reporter gene in HEK293 cells transiently transfected with one or more TLRs. Results demonstrated that C. sordellii was well recognized by cells expressing TLR2 alone (RLU ¼ 0.95; Fig. 1). This response was nearly 2-fold higher when TLR2 was co-expressed with TLR6 (RLU ¼ 1.89). In contrast, C. sordellii-induced NF-kB activation was significantly decreased in cells expressing the TLR2/1 heterodimer (RLU ¼ 0.4). Minimal TLR4mediated NF-kB activation was elicited by C. sordellii (RLU ¼ 0.08) and the co-expression of TLR2/4 generated responses comparable to levels obtained with TLR2 alone (RLU ¼ 1.1), confirming the absence of contaminating LPS in these bacterial preparations. The intensity of the C. sordellii-induced TLR2-mediated responses (alone or co-expressed with other TLRs) was dependent on the bacterial dose used for challenge (Fig. 2).

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C. sordellii LPS

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* *

1.0

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0.5

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0 TLR4

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TLR2/4

No Treatment

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Fig. 1. C. sordellii and LPS strongly stimulates NF-kB activation through TLR receptors in HEK293 cells. HEK293 cells expressing TLR 1,2, 4, or 6 alone or in combination were stimulated for 4 h with either nothing, LPS (10 ng/mL) or washed, LPS-free, heat killed C. sordellii at an OD600 nm ¼ 0.3. Activation of NF-kB was determined through an ELAMdependent luciferase reporter system as described in Section 2. Data are reported as the IL-1b-normalized ratio of ELAM-luciferase/Renilla luciferase activities. *P < 0.05 and **P < 0.001 compared to TLR2 by one way ANOVA with Tukeys test. Experiment was performed in triplicate on three occasions.

3.2. C. sordellii stimulates cytokine production by human mononuclear cells Subsequent experiments investigated whether C. sordellii stimulated cytokine production by human mononuclear cells. For this purpose, peripheral blood mononuclear cells (PBMC) were isolated from human whole blood and exposed to washed, heat-killed C. sordellii for 16 h. TNFa and IL-10 in the overlying culture media were measured by commercial ELISA. Results demonstrated that monocytes exposed to C. sordellii generated amounts of TNFa that were comparable to that stimulated by the LPS control (19.1  2.13 ng/mL and 19.3  3.56 ng/mL respectively; Fig. 3a). Stimulation of mononuclear cells with C. sordellii also induced production of the immunoregulatory cytokine IL-10 (50.61  4.03 ng/ml; Fig. 3b), again at levels similar to that induced by LPS (56.28  6.13 ng/mL).

No Treatment OD600 = 0.06 OD600 = 0.30

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40 30 20 10 0 No Treatment

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Fig. 3. C. sordellii induces inflammatory-associated cytokine production in human mononuclear cells. Human monocytes (2  106/well) were stimulated for 16 h with either sterile saline, LPS (10 ng/mL) or washed, LPS-free heat-killed C. sordellii at OD600nm ¼ 0.014. Following exposure, culture supernatants were collected and TNFa (a) and IL-10 (b) were measured in duplicate by commercial ELISA. *P < 0.001 compared to No Treatment control determined by one way ANOVA with Tukeys test.

To confirm the functional role of TLR2 in C. sordellii-induced TNFa production by PBMCs, blocking experiments utilizing antibodies against TLR2 and TLR4 were performed. Antibody to TLR2, but not anti-TLR4 antibody, reduced C.sordellii-induced TNFa production by w30% (Fig. 4). In contrast, anti-TLR4, but not antiTLR2, antibody significantly reduced the LPS-induced TNFa response (Fig. 4).

OD600 = 1.50 2.5

3.3. TLR usage patterns of other clostridial pathogens 2.0 1.5 1.0 0.5 0.0 TLR2

TLR4

TLR2/1

TLR2/4

TLR2/6

Fig. 2. C. sordellii dose-dependently activates NF-kB signaling in TLR-expressing HEK293 cells. HEK293 cells expressing TLR 1,2, 4, or 6 alone or in combination were stimulated for 4 h with either nothing or washed, heat killed clostridial species at OD600nm ¼ 0.06, 0.30 or 1.50. Activation of NFkB was determined through an ELAMdependent luciferase reporter system as in Fig. 1. Data are given as the IL-1bnormalized ratio of ELAM-luciferase/Renilla luciferase activities. Experiment was performed in triplicate on three occasions.

To investigate TLR utilization in recognition of other pathogenic clostridial species, HEK293 cells expressing TLRs (individually and in combination) were exposed to C. perfringens, C. septicum (both cause gas gangrene [17,18]), C. clostridioforme (causes osteomyelitis and abscesses-related infections [19,20]), or C. bolteae (recently associated with late-onset autism [21]). All species were well recognized by TLR2 alone (Fig. 5) and engagement of TLR2/6 increased the responses. As observed with all clostridium species, C. septicum was well recognized by TLR2 alone and with greater intensity when co-expressed with TLR6 (RLU ¼ 2.75 and 3.40 respectively; Fig. 5). Unexpectedly, C. septicum stimulated marked NF-kB activation in transfected HEK293 cells irrespective of the TLRs expressed (Fig. 5). Subsequent studies demonstrated that nonTLR-transfected HEK293 cells also respond to C. septicum (data not shown), likely via the interaction of the organisms flagella with

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TNF-alpha (ng/mL)

20

15

(15.2–25.5 ng/mL). These data paralleled IL-8 production by TLRtransfected HEK293 cells exposed to heat-killed clostridium organisms. For example, C. sordellii generated TLR2 and TLR2/6 RLU values of 0.95 and 1.89 (Fig. 5) and IL-8 measurement of 73.2 and 121.6 pg/mL, while C. perfringens had TLR2 and TLR2/6 RLU values of 1.27 and 2.42 (Fig. 5) and IL-8 measurements of 55.7 and 189.5 pg/mL, respectively.

No Antibody IgG Isotype anti-TLR2 anti-TLR4 γ

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4. Discussion

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LPS

C. sordellii

Fig. 4. TNFa production by isolated human mononuclear cells is blocked by anti-TLR2 and anti-TLR4 antibodies. Human peripheral blood mononuclear cells (2  106/well) were isolated from human whole blood and pre-exposed to either anti-TLR2 mAb (20 mg/mL), anti-TLR4 mAb or isotype matched control IgG mAb (20 mg/mL) and treated with either nothing, washed, LPS-free heat-killed C. sordellii at OD600nm ¼ 0.014, or LPS (10 ng/mL). Following treatment, culture supernatants were collected and TNFa was measured in duplicate by commercial ELISA. *P < 0.05 and g ¼ P ¼ 0.07 compared to the No Antibody control for each respective treatment, determined by one way ANOVA with Tukeys test.

TLR5 which is constitutively expressed by HEK293 cells (unpublished observations). Co-expression of TLR2/1 significantly decreased the responses to C. perfringens and C. clostridioforme but enhanced the response to C. bolteae (Fig. 5). No TLR4-dependent signaling was observed for any of the species tested and TLR2/4 co-expression responses were nearly identical to that of TLR2 alone for all species (Fig. 4).

3.4. Cytokine induction by human monocytes following Clostridium spp. exposure All clostridial species elicited TNFa production by isolated human mononuclear cells, but with differing potencies

NF-κB Activation (IL-1 Normalized RLU)

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perfringens sordellii septicum bolteae clostridioforme

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Fig. 5. TLR-dependent innate immune recognition of the clostridia is species dependent. HEK293 cells were transfected with TLRs 1, 2, 4, or 6, alone or in combination and stimulated for 4 h with either sterile saline or LPS-free, washed heat killed clostridia at OD600nm ¼ 0.3. Activation of NF-kB is given as the IL-1b-normalized ratio of ELAMluciferase/Renilla luciferase activities. *P < 0.05 and **P < 0.001 compared to TLR2 by one way ANOVA with Tukeys test. Statistical comparisons were performed individually for each clostridium species. The experiment was performed in triplicate on three occasions with the C. perfringens, C. sordellii, and C. septicum and once with C. bolteae and C. clostridioforme. Marked C. septicum NF-kB activation reflects interaction of the organisms flagella with TLR5 which is constitutively expressed by HEK293 cells.

Six distinctive clinical features uniquely characterize infections due to C. sordellii: a marked leukocytosis termed the leukemoid reaction, refractory hypotension, severe tachycardia, profound capillary leak syndrome, hemoconcentration and a persistent lack of fever [1]. The absence of fever and only limited signs of inflammation (e.g., redness, pain) at the infection site confound and delay diagnosis and thereby contribute to mortality rates of 60–100% in C. sordellii infection. Whats more, they suggest a species-specific, systemic dysfunction of the innate immune response since soft tissue infection with other histotoxic clostridial species are met with a robust febrile response and severe pain at the infection site. Understanding the mechanisms responsible for the unique immune dysfunction in C. sordellii infection could shed light on the pathogenesis of this devastating infection and could suggest targets for diagnosis or intervention. Because the innate immune response is the bodys first line of defense against invading pathogens, we first investigated whether intact C. sordellii bacilli were properly recognized by Toll-like receptors (TLRs). TLRs are key players in innate immunity and host defense [22,23]. During infection, TLRs bind shared microbial ligands and elicit the expression of several pro-inflammatory cytokines including TNFa, interleukin (IL)-1, interferon gamma (INF-g) [24]. These inflammatory mediators trigger a cascade of signaling events resulting in a highly orchestrated inflammatory response at both the local and systemic levels. Of the 11 TLRs recognized in humans, a critical role for TLR2 in the recognition of some Gram-positive pathogens such as Staphylococcus aureus [25–28], Streptococcus pyogenes [29,30] and Streptococcus pneumoniae [31] has been demonstrated in vitro and in vivo. Using an ELAM-NF-kB luciferase reporter system in TLR-transfected HEK cells, we demonstrate for the first time that TLR2 also mediates the recognition of several clinically important clostridial species, including C. sordellii. Further, the robustness of the TLR2mediated response to C. sordellii was comparable to that of other pathogenic clostridia and to that elicited by LPS. We next investigated whether engagement of TLRs elicits a functional response in human monocytes. Indeed, our data also demonstrate that engagement of the mononuclear cell TLR system by the various clostridia, including C. sordellii, results in significant TNFa production. Since TNFa and IL-1b are endogenous pyrogens [32], our findings suggest that the absence of fever in patients with C. sordellii infection cannot be attributed to failure of the innate immune system to recognize the pathogen or to signal downstream activational events. Specificity in TLR2-mediated pathogen recognition arises through combinatorial association with either TLR1 or TLR6 [33–35]. For example, diacylated lipoproteins require the heterodimer TLR2/ 6 for recognition, whereas triacylated lipoproteins induce NF-kB signaling mainly through TLR2/1 [36,37]. All clostridial species in this study generated maximal NF-kB activation in transfected cells co-expressing TLRs 2 and 6 (except C. bolteae whose TLR2/6 and TLR2/1 responses were equivalent), suggesting a relative abundance of diacylated lipoproteins in the clostridial cell wall. The inability of anti-TLR2 to completely inhibit TNFa production by C. sordellii-stimulated monocytes supports the notion that

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recognition of the clostridia is optimal following engagement of TLR2/6 heterodimers, though other as yet unidentified receptors may also play important roles. What, then, accounts for the characteristic lack of fever in C. sordellii infections? Because C. sordellii also stimulated production of the anti-inflammatory mediator IL-10 in vitro, it is possible that the unique clinical manifestations of this infection reflect an imbalance in the in vivo production of critical immune mediators. Alternatively, because our system utilized washed, heat-killed organisms, it is possible that exotoxins produced specifically by C. sordellii disrupt TLR-mediated signaling under active infection conditions. C. sordellii produces two glycosyltransferases, lethal toxin (LT) and hemorrhagic toxin (HT), which modify small GTPases of the Rho signaling family [38]. A role for Rho and Rho-like GTPases (e.g., Rac-1) in TLR2-mediated NF-kB activation has been reported [39,40]. Studies in our laboratory showed that pre-treatment of cultured adrenal cells with C. sordellii LT and HT blocks LPS-induced glucocorticoid production (unpublished data) – a process inducible by TLR engagement [41] and mediated by Rho-dependent signaling [42]. Investigations of the direct effects of these, and other C. sordellii toxins, on TLR-mediated cytokine production are currently ongoing in our laboratory. 5. Conclusion In conclusion, the mechanisms responsible for the unique clinical features of C. sordellii infection are, at best, vaguely understood. However, the relationship between the development of these features and fatal outcome is clear. Data reported in this study demonstrate that cells of the innate immune system can recognize and respond to C. sordellii and suggest that potent exotoxins uniquely produced by this pathogen can derail this process. A better understanding of this host/pathogen interaction is needed to provide vital diagnostic tools and novel therapeutic strategies to reduce the morbidity and mortality associated with C. sordellii infection. Acknowledgements This material is based upon work supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs Service, and by a Centers of Biomedical Research Excellence (COBRE) grant from the National Institutes of Health (NCRR P20RR15587). Portions of this work were presented in abstract form at the 8th biennial meeting of the Anaerobe Society of the Americas, Boise, Idaho, July 2006. The presented information received the Finegold Award for best abstract. References [1] Aldape MJ, Bryant AE, Stevens DL. Clostridium sordellii infection: epidemiology, clinical findings, and current perspectives on diagnosis and treatment. Clin Infect Dis 2006 Dec 1;43(11):1436–46. Epub 2006 Oct 30. [2] Brett MM, Hood J, Brazier JS, Duerden BI, Hahne´ SJ. Soft tissue infections caused by spore-forming bacteria in injecting drug users in the United Kingdom. Epidemiol Infect 2005 Aug;133(4):575–82. [3] Kimura AC, Higa JI, Levin RM, Simpson G, Vargas Y, Vugia DJ. Outbreak of necrotizing fasciitis due to Clostridium sordellii among black-tar heroin users. Clin Infect Dis 2004 May 1;38(9):e87–91. Epub 2004 Apr 14. [4] Fischer M, Bhatnagar J, Guarner J, Reagan S, Hacker JK, Van Meter SH, et al. Fatal toxic shock syndrome associated with Clostridium sordellii after medical abortion. N Engl J Med 2005 Dec 1;353(22):2352–60. [5] Murray S, Wooltorton E. Septic shock after medical abortions with mifepristone (Mifeprex, RU 486) and misoprostol. CMAJ 2005 Aug 30;173(5):485. Epub 2005 Aug 10. [6] Wiebe E, Guilbert E, Jacot F, Shannon C, Winikoff B. A fatal case of Clostridium sordellii septic shock syndrome associated with medical abortion. Obstet Gynecol 2005 Mar;105(3):673.

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[7] Dasari P, Nicholson IC, Zola H. Toll-like receptors. J Biol Regul Homeost Agents 2008 Jan–Mar;22(1):17–26. [8] Underhill DM, Ozinsky A. Toll-like receptors: key mediators of microbe detection. Curr Opin Immunol 2002 Feb;14(1):103–10. [9] Ghosh S, Hayden MS. New regulators of NF-kappaB in inflammation. Nat Rev Immunol 2008 Nov;8(11):837–48. [10] Lorenz E. TLR2 and TLR4 expression during bacterial infections. Curr Pharm Des 2006;12(32):4185–93. [11] Harrison LM, Rallabhandi P, Michalski J, Zhou X, Steyert SR, Vogel SN, et al. Vibrio cholerae flagellins induce Toll-like receptor 5-mediated interleukin-8 production through mitogen-activated protein kinase and NF-kappaB activation. Infect Immun 2008 Dec;76(12):5524–34. Epub 2008 Sep 22. [12] Song Y, Liu C, Molitoris DR, Tomzynski TJ, Lawson PA, Collins MD, et al. Clostridium bolteae sp. nov., isolated from human sources. Syst Appl Microbiol 2003 Mar;26(1):84–9. [13] Aldape MJ, Bryant AE, Ma Y, Stevens DL. The leukemoid reaction in Clostridium sordellii infection: neuraminidase induction of promyelocytic cell proliferation. J Infect Dis 2007 Jun 15;195(12):1838–45. Epub 2007 May 2. [14] Hajjar AM, OMahony DS, Ozinsky A, Underhill DM, Aderem A, Klebanoff SJ, et al. Cutting edge: functional interactions between toll-like receptor (TLR) 2 and TLR1 or TLR6 in response to phenol-soluble modulin. J Immunol 2001 Jan 1;166(1):15–9. [15] Chen C, Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 1987 Aug;7(8):2745–52. [16] Hackett SP, Stevens DL. Streptococcal toxic shock syndrome: synthesis of tumor necrosis factor and interleukin-1 by monocytes stimulated with pyrogenic exotoxin A and streptolysin O. J Infect Dis 1992 May;165(5):879–85. [17] Stevens DL, Bryant AE. The role of clostridial toxins in the pathogenesis of gas gangrene. Clin Infect Dis 2002 Sep 1;35(Suppl. 1):S93–100. [18] Dylewski J, Drummond R, Rowen J. A case of Clostridium septicum spontaneous gas gangrene. CJEM 2007 Mar;9(2):133–5. [19] Spitzer RD, Ratzan KR. Chronic osteomyelitis due to Clostridium clostridiiforme. South Med J 1991 May;84(5):671–2. [20] Elsayed S, Zhang K. Human infection caused by Clostridium hathewayi. Emerg Infect Dis 2004 Nov;10(11):1950–2. [21] Song Y, Liu C, Finegold SM. Real-time PCR quantitation of clostridia in feces of autistic children. Appl Environ Microbiol 2004 Nov;70(11):6459–65. [22] Beutler B. Toll-like receptors: how they work and what they do. Curr Opin Hematol 2002 Jan;9(1):2–10. [23] Janeway Jr CA, Medzhitov R. Innate immune recognition. Annu Rev Immunol 2002;20:197–216. Epub 2001 Oct 4. [24] Gabay C, Kushner I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med 1999 Feb 11;340(6):448–54. [25] Zhao YT, Guo JH, Wu ZL, Xiong Y, Zhou WL. Innate immune responses of epididymal epithelial cells to Staphylococcus aureus infection. Immunol Lett 2008 Aug 15;119(1–2):84–90. Epub 2008 Jun 4. [26] Li Q, Kumar A, Gui JF, Yu FS. Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2. Microb Pathog 2008 May;44(5):426–34. Epub 2007 Nov 28. [27] Hoebe K, Georgel P, Rutschmann S, Du X, Mudd S, Crozat K, et al. CD36 is a sensor of diacylglycerides. Nature 2005 Feb 3;433(7025):523–7. [28] Takeuchi O, Hoshino K, Akira S. Cutting edge: TLR2-deficient and MyD88deficient mice are highly susceptible to Staphylococcus aureus infection. J Immunol 2000 Nov 15;165(10):5392–6. [29] Miettinen M, Veckman V, Latvala S, Sareneva T, Matikainen S, Julkunen I. Live Lactobacillus rhamnosus and Streptococcus pyogenes differentially regulate Toll-like receptor (TLR) gene expression in human primary macrophages. J Leukoc Biol 2008 Oct;84(4):1092–100. Epub 2008 Jul 14. [30] Loof TG, Goldmann O, Medina E. Immune recognition of Streptococcus pyogenes by dendritic cells. Infect Immun 2008 Jun;76(6):2785–92. Epub 2008 Apr 7. [31] Xu F, Droemann D, Rupp J, Shen H, Wu X, Goldmann T, et al. Modulation of the inflammatory response to Streptococcus pneumoniae in a model of acute lung tissue infection. Am J Respir Cell Mol Biol 2008 Nov;39(5):522–9. Epub 2008 May 15. [32] Dinarello CA, Cannon JG, Wolff SM, Bernheim HA, Beutler B, Cerami A, et al. Tumor necrosis factor (cachectin) is an endogenous pyrogen and induces production of interleukin 1. J Exp Med 1986 Jun 1;163(6):1433–50. [33] Ozinsky A, Underhill DM, Fontenot JD, Hajjar AM, Smith KD, Wilson CB, et al. The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. Proc Natl Acad Sci USA 2000 Dec 5;97(25):13.766–71. [34] Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, et al. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 1999 Oct;11(4):443–51. [35] Takeuchi O, Kawai T, Sanjo H, Copeland NG, Gilbert DJ, Jenkins NA, et al. TLR6: a novel member of an expanding toll-like receptor family. Gene 1999 Apr 29;231(1–2):59–65. [36] Takeuchi O, Kawai T, Mu¨hlradt PF, Morr M, Radolf JD, Zychlinsky A, et al. Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int Immunol 2001 Jul;13(7):933–40. [37] Takeuchi O, Sato S, Horiuchi T, Hoshino K, Takeda K, Dong Z, et al. Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J Immunol 2002 Jul 1;169(1):10–4. [38] Genth H, Hofmann F, Selzer J, Rex G, Aktories K, Just I. Difference in protein substrate specificity between hemorrhagic toxin and lethal toxin from Clostridium sordellii. Biochem Biophys Res Commun 1996 Dec 13;229(2):370–4.

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M.J. Aldape et al. / Anaerobe 16 (2010) 125–130

[39] Montaner S, Perona R, Saniger L, Lacal JC. Multiple signalling pathways lead to the activation of the nuclear factor kappaB by the Rho family of GTPases. J Biol Chem 1998 May 22;273(21):12,779–12,785. [40] Arbibe L, Mira JP, Teusch N, Kline L, Guha M, Mackman N, et al. Toll-like receptor 2-mediated NF-kappa B activation requires a Rac1-dependent pathway. Nat Immunol 2000 Dec;1(6):533–40.

[41] Vakharia K, Hinson JP. Lipopolysaccharide directly stimulates cortisol secretion by human adrenal cells by a cyclooxygenase-dependent mechanism. Endocrinology 2005 Mar;146(3):1398–402. Epub 2004 Nov 24. [42] Otis M, Gallo-Payet N. Role of MAPKs in angiotensin II-induced steroidogenesis in rat glomerulosa cells. Mol Cell Endocrinol 2007 Feb;265-266:126–30. Epub 2007 Jan 9.