## THE IMMUNOLOGY OF SEPSIS – UNDERSTANDING HOST SUSCEPTIBILITY, PATHOGENESIS OF DISEASE, AND AVENUES FOR FUTURE TREATMENT

EDITED BY : Luregn J. Schlapbach, Johannes Trück and Thierry Roger PUBLISHED IN : Frontiers in Immunology, Frontiers in Microbiology and Frontiers in Medicine

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ISSN 1664-8714 ISBN 978-2-88963-939-7 DOI 10.3389/978-2-88963-939-7

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## THE IMMUNOLOGY OF SEPSIS – UNDERSTANDING HOST SUSCEPTIBILITY, PATHOGENESIS OF DISEASE, AND AVENUES FOR FUTURE TREATMENT

Topic Editors:

Luregn J. Schlapbach, The University of Queensland, Australia Johannes Trück, University Children's Hospital Zurich, Switzerland Thierry Roger, Centre Hospitalier Universitaire Vaudois (CHUV), Switzerland

We acknowledge the initiation and support of this Research Topic by the International Union of Immunological Societies (IUIS). We hereby state publicly that the IUIS has had no editorial input in articles included in this Research Topic, thus ensuring that all aspects of this Research Topic are evaluated objectively, unbiased by any specific policy or opinion of the IUIS.

Citation: Schlapbach, L. J., Trück, J., Roger, T., eds. (2020). The Immunology of Sepsis – Understanding Host Susceptibility, Pathogenesis of Disease, and Avenues for Future Treatment. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88963-939-7

# Table of Contents

Cassiano Felippe Gonçalves-de-Albuquerque, Ina Rohwedder, Adriana Ribeiro Silva, Alessandra Silveira Ferreira, Angela R. M. Kurz, Céline Cougoule, Sarah Klapproth, Tanja Eggersmann, Johnatas D. Silva, Gisele Pena de Oliveira, Vera Luiza Capelozzi, Gabriel Gutfilen Schlesinger, Edlaine Rijo Costa, Rita de Cassia Elias Estrela Marins, Attila Mócsai, Isabelle Maridonneau-Parini, Barbara Walzog, Patricia Rieken Macedo Rocco, Markus Sperandio and Hugo Caire de Castro-Faria-Neto

*26 The Surface-Exposed Protein SntA Contributes to Complement Evasion in Zoonotic* Streptococcus suis

Simin Deng, Tong Xu, Qiong Fang, Lei Yu, Jiaqi Zhu, Long Chen, Jiahui Liu and Rui Zhou

Anina Schneider, Manuela Weier, Jacobus Herderschee, Matthieu Perreau, Thierry Calandra, Thierry Roger and Eric Giannoni

	- Jan G. Zijlstra, Matijs van Meurs and Jill Moser

Bastiaan W. Haak, Hallie C. Prescott and W. Joost Wiersinga

*97 "Real-Time" High-Throughput Drug and Synergy Testing for Multidrug-Resistant Bacterial Infection: A Case Report* Wei Sun, Shayla Hesse, Miao Xu, Richard W. Childs, Wei Zheng and Peter R. Williamson

*102 Prophylactic Treatment With Simvastatin Modulates the Immune Response and Increases Animal Survival Following Lethal Sepsis Infection* Jose A. F. Braga Filho, Afonso G. Abreu, Carlos E. P. Rios, Liana O. Trovão,

Dimitri Luz F. Silva, Dalila N. Cysne, Johnny R. Nascimento, Thiare S. Fortes, Lucilene A. Silva, Rosane N. M. Guerra, Márcia C. G. Maciel, Carlos H. Serezani and Flávia R. F. Nascimento

Chloé Albert-Vega, Dina M. Tawfik, Sophie Trouillet-Assant, Laurence Vachot, François Mallet and Julien Textoris

Cennan Yin, Chenyun Wu, Xinyue Du, Yan Fang, Juebiao Pu, Jianhua Wu, Lili Tang, Wei Zhao, Yongqiang Weng, Xiaokui Guo, Guangjie Chen and Zhaojun Wang

*169 Sepsis Induces a Long-Lasting State of Trained Immunity in Bone Marrow Monocytes*

Katharina Bomans, Judith Schenz, Isabella Sztwiertnia, Dominik Schaack, Markus Alexander Weigand and Florian Uhle

*180 Protection Against Invasive Infections in Children Caused by Encapsulated Bacteria*

Manish Sadarangani

*189 Resveratrol-Mediated Attenuation of* Staphylococcus aureus *Enterotoxin B-Induced Acute Liver Injury is Associated With Regulation of microRNA and Induction of Myeloid-Derived Suppressor Cells*

Sabah Kadhim, Narendra P. Singh, Elizabeth E. Zumbrun, Taixing Cui, Saurabh Chatterjee, Lorne Hofseth, Abduladheem Abood, Prakash Nagarkatti and Mitzi Nagarkatti

*201 Macrophage Activation-Like Syndrome: A Distinct Entity Leading to Early Death in Sepsis*

Eleni Karakike and Evangelos J. Giamarellos-Bourboulis

*211 Ferritin Light Chain Confers Protection Against Sepsis-Induced Inflammation and Organ Injury*

Abolfazl Zarjou, Laurence M. Black, Kayla R. McCullough, Travis D. Hull, Stephanie K. Esman, Ravindra Boddu, Sooryanarayana Varambally, Darshan S. Chandrashekar, Wenguang Feng, Paolo Arosio, Maura Poli, Jozsef Balla and Subhashini Bolisetty

## *226 Myeloid-Derived Suppressor Cells in Sepsis*

Irene T. Schrijver, Charlotte Théroude and Thierry Roger

*236 Monocyte HLA-DR Assessment by a Novel Point-of-Care Device is Feasible for Early Identification of ICU Patients With Complicated Courses—A* Proof-of-Principle *Study* Sandra Tamulyte, Jessica Kopplin, Thorsten Brenner, Markus Alexander Weigand and Florian Uhle *247 The Long Pentraxin PTX3 as a Humoral Innate Immunity Functional Player and Biomarker of Infections and Sepsis* Rémi Porte, Sadaf Davoudian, Fatemeh Asgari, Raffaella Parente, Alberto Mantovani, Cecilia Garlanda and Barbara Bottazzi *258 Challenge to the Intestinal Mucosa During Sepsis* Felix Haussner, Shinjini Chakraborty, Rebecca Halbgebauer and Markus Huber-Lang *274 Evaluation of Mannose Binding Lectin Gene Variants in Pediatric Influenza Virus-Related Critical Illness* Emily R. Levy, Wai-Ki Yip, Michael Super, Jill M. Ferdinands, Anushay J. Mistry, Margaret M. Newhams, Yu Zhang, Helen C. Su, Gwenn E. McLaughlin, Anil Sapru, Laura L. Loftis, Scott L. Weiss, Mark W. Hall, Natalie Cvijanovich, Adam Schwarz, Keiko M. Tarquinio, Peter M. Mourani, PALISI PICFLU Investigators and Adrienne G. Randolph *286 Epigenetics in Sepsis: Understanding Its Role in Endothelial Dysfunction, Immunosuppression, and Potential Therapeutics*

Deborah Cross, Ruth Drury, Jennifer Hill and Andrew J. Pollard

# Editorial: The Immunology of Sepsis—Understanding Host Susceptibility, Pathogenesis of Disease, and Avenues for Future Treatment

Luregn J. Schlapbach1,2 \* †‡, Johannes Trück 2†‡ and Thierry Roger 3†‡

1 Paediatric Critical Care Research Group, Paediatric Intensive Care Unit, Child Health Research Centre, Queensland Children's Hospital, The University of Queensland, Brisbane, QLD, Australia, 2 University Children's Hospital Zurich and University of Zurich, Zurich, Switzerland, 3 Lausanne University Hospital and University of Lausanne, Epalinges, Switzerland

Keywords: immune response, infection, organ dysfunction, personalized medicine, sepsis, septic shock, dysregulated host response

Edited and reviewed by: Ian Marriott,

University of North Carolina at Charlotte, United States

> \*Correspondence: Luregn J. Schlapbach<EMAIL_ADDRESS>
### †ORCID:

Luregn J. Schlapbach orcid.org/0000-0003-2281-2598 Johannes Trück orcid.org/0000-0002-0418-7381 Thierry Roger orcid.org/0000-0002-9358-0109

‡These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology

Received: 02 May 2020 Accepted: 19 May 2020 Published: 23 June 2020

#### Citation:

Schlapbach LJ, Trück J and Roger T (2020) Editorial: The Immunology of Sepsis—Understanding Host Susceptibility, Pathogenesis of Disease, and Avenues for Future Treatment. Front. Immunol. 11:1263. doi: 10.3389/fimmu.2020.01263 **Editorial on the Research Topic**

### **The Immunology of Sepsis—Understanding Host Susceptibility, Pathogenesis of Disease, and Avenues for Future Treatment**

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection (1). Most recent estimations suggest that sepsis affects about 49 million people and accounts for around 11 million deaths each year worldwide (2), making it one of the leading causes of preventable death in all age groups. Sepsis is a health priority according to the World Health organization (3), which provided recommendations to improve the prevention, diagnosis and management of sepsis. Although sepsis mortality has decreased over the past two decades (4, 5), the incidence of sepsis continues to rise due to a higher number of patients with complex conditions such as immunosuppression as well as an increase in more elderly people. A basic requirement for effective sepsis management is timely and appropriate antimicrobial therapy (6, 7). With the increase of infections caused by multidrug resistant bacteria, the advances made toward rapid multidrug testing to enable personalized antimicrobial therapies is promising (Sun et al.). However, a major obstacle to improving sepsis outcomes is the lack of knowledge on the intricate interplay between host defense, infection, and pathogen virulence, as well as timing and nature of interventions. Over the past decade, our understanding of sepsis has evolved from the earlier concept of sepsis as a result of an excessive inflammatory response to the current notion that sepsis outcomes are more likely related to the dynamic balance between pro-inflammatory and counteracting anti-inflammatory mechanisms. To this end, animal models and a number of human observational studies have paved the way toward precision trials on immunotherapy for sepsis (Zijlstra et al.).

The aim of the Frontiers in Immunology Research Topic "The Immunology of Sepsis–Understanding Host Susceptibility, Pathogenesis of Disease, and Avenues for Future Treatment" was to collect state-of-the art articles and reviews on the role of the host immune system affecting susceptibility, presentation, and outcome of sepsis. We hereby provide an overview of this Frontiers in Immunology topic which includes 11 original articles, 13 review articles, 1 commentary, and 1 case report.

**6**

### SUSCEPTIBILITY TO SEPSIS

Sepsis susceptibility may result from maturational, genetic, or acquired alterations of the immune system. The incidence of sepsis is highest in newborns (8), and an increasing body of studies characterize distinct patterns of immune responses in this age group compared to children and adults. Schneider et al. for example, demonstrate that neonatal macrophages express substantially less tumor necrosis factor compared to adult cells, regulated through the transcription factor interferon regulatory factor 5 (IRF5).

Considering how few patients develop sepsis in comparison to the exposed population, there is biological plausibility and epidemiologic evidence for underlying genetic mechanisms affecting susceptibility to sepsis (9, 10). These may affect both very rare variants associated with extreme phenotypes (11) and common variants that may be of relevance at the populational level (12). Deficiency in mannose-binding lectin (MBL) emerged two decades ago as a promising candidate to investigate this hypothesis (13). Although polymorphisms affecting MBL serum levels are common and the role of complement in host defenses against bacterial infections is well known, previous reports on the relevance of MBL deficiency in susceptibility to infection remained conflicting (14). Using a well-phenotyped prospective intensive care unit cohort, Levy et al., genotyped 420 pediatric patients with influenza-associated organ dysfunction for variants in the MBL2 gene expected to result in low serum MBL levels. No clear relationship was observed between genetic variants and overall outcomes in the cohort, neither in within-cohort, nor in trio or control analyses. Interestingly, and similar to a previous study, low-MBL producing variants were more common in a subset of fatal cases with methicillin-resistant Staphylococcus aureus (MRSA), but the relevance of this finding remains to be confirmed in larger studies.

### HOST-PATHOGEN INTERACTION

Children are prone to severe infections caused by encapsulated bacteria such as Streptococcus pneumoniae, Streptococcus agalactiae (Group B streptococcus, GBS), Neisseria meningitis and Haemophilus influenzae. The polysaccharide capsule is a key bacterial virulence factor hindering opsonophagocytosis and complement-mediated bacteriolysis. However, it also represents an effective target for vaccine development. In his review, Sadarangani describes the mechanisms of vaccine-induced protection and illustrates successful examples of pneumococcal and meningococcal capsular conjugate vaccines, while challenges remain in the development of effective vaccines against non-type b H. influenzae and GBS that also carry polysaccharide capsules. The article by Kadhim et al., demonstrates a novel potential drug candidate to block staphylococcus enterotoxin-induced organ damage. Finally, Deng et al. investigate mechanisms of complement evasion in streptococcal strains.

Very timely considering the current coronavirus disease 2019 (COVID-19) pandemic, and giving credit to an often overlooked problem, Lin et al., provide a thorough overview of the epidemiology and pathogenesis of viral sepsis with a focus on herpes simplex, influenza, and dengue viruses, as well as entero- and parechoviruses. Recognition of a viral origin of sepsis could allow targeted treatment and reduce the unnecessary use of antibiotics.

### UNDERSTANDING THE DYSREGULATED HOST RESPONSE TO INFECTION

While the concept of a "dysregulated host response to infection leading to organ dysfunction" in sepsis is broadly accepted, our understanding of the underlying mechanisms remains very limited. The contributions to this Frontiers topic shed light on a number of pathways that are likely involved.

Previous studies have shown that a subgroup of septic children and adults reveal similar patterns to patients with hemophagocytic lymphohistocytosis (HLH), or macrophage activation syndrome (MAS) such as cytokine storm, hyperferritinemia, and multi-organ dysfunction. This entity has been termed macrophage activation-like syndrome (MALS). Karakike et al. provide an overview of the available evidence on this topic, while the results of the first randomized clinical trials (RCTs) on MALS are eagerly awaited. Zarjou et al. assessed the inhibitory role of myeloid ferritin heavy chain and ferritin light chain in a mouse model on nuclear factor kappa-light-chainenhancer of activated B cells (NF-kB) activation, illustrating a potential immunomodulatory role of ferritin light chain.

More recently, insight into pro- and anti-inflammatory responses to the exposure of pathogen- and damage-associated molecular patterns (PAMPs and DAMPs) suggest a dynamic and heterogenous process (15). A fascinating research area in this regard relates to epigenetic processes affecting gene expression during sepsis. Cross et al. provide an overview on the literature on this topic. They summarize the rapidly growing number of mainly laboratory studies indicating that epigenetic mechanisms are thoroughly perturbed during sepsis, and are related to endothelial dysfunction and immunosuppression. These observations may open up new treatment options, such as drug interventions with histone deacetylase inhibitors for which pre-clinical animal studies suggest a potential benefit. It will be interesting to see whether candidate drugs progress to clinical studies on immune modulation in sepsis in the coming decade. Another possible drug approach that could support a more balanced immune response is the blocking of the Src family of tyrosine kinases with dasatinib, a drug for the treatment of chronic myeloid leukemia and acute lymphoblastic leukemia. Indeed, Gonçalves-de-Albuquerque et al. report promising results of using dasatinib in a mouse model of polymicrobial sepsis.

Several other proteins and cells represent both encouraging biomarkers and therapeutic targets for future strategies to combat sepsis. For example, Schrijver et al. report that myeloid-derived suppressor cells (MDSCs) are a heterogenous group of immature cells that expand in a number of conditions including sepsis. MDSCs suppress immune responses of different cell types in the early and late phases of sepsis, and pre-clinical studies show distinct patterns of expansion of MDSC subpopulations Observational studies have already reported an association between high proportions of blood MDSCs and poor outcome in sepsis patients. In a mouse model, Bomans et al. have elucidated mechanisms involved in the functional reprogramming of naïve bone marrow monocytes after sepsis, which lead to a "memory" of the innate immune system.

Other articles submitted to this Research Topic illustrate a number of recent discoveries of cells and pathways that contribute to the host response to sepsis. Yin et al., demonstrate that phosphatase regenerating liver 2 (PRL2) regulates the generation of reactive oxygen species in macrophages and thereby contributes to bactericidal activity. Sjaastad et al., assessed the response to polymicrobial sepsis using a mouse model and observed features consistent with chronic immunoparalysis, measured by reduced antigen-specific T cell-dependent B cell responses.

Although the intestinal system has not been commonly considered in organ dysfunction scores (1), it represents an extraordinary body surface area containing a high density of lymphatic tissues and immune cells. The regulation between the host immune system and intestinal pathogens as well as bacterial translocation play a key role in the dynamic processes during and after shock. Increasing our understanding of these events may point to ways for secondary interventions, as summarized in the review by Haussner et al., on the role of the intestinal mucosa during sepsis. Expanding from this work, the importance of intestinal microbiota in sepsis is explored in the excellent review by Haak et al. shedding light on the therapeutic potential of microbiome-modifying strategies for prevention and treatment of sepsis and sepsis-related late mortality.

### BIOMARKERS, MONITORING THE IMMUNE SYSTEM, AND THE FUTURE OF IMMUNOMODULATION IN SEPSIS

The immune status of patients with sepsis is subject to rapid progression, and hyperinflammation and immunoparalysis may change dynamically. The failures to characterize the predominant immunological phenotype of sepsis patients coupled with considerable disease and patient heterogeneity represent major obstacles to develop effective immunomodulation-based trials. Peters van Ton et al. discuss the critical need for accurate monitoring of associated immune dysregulation to select patients more likely to benefit from targeted immunomodulatory interventions, and to facilitate enrolment in clinical studies toward personalized medicine.

The initial response to infection includes activation of innate immunity through soluble and cell-bound pattern recognition receptors. Porte et al. for example, review the available evidence on the role of the long pentraxin 3 (PTX3) in sepsis, a molecule with high homology with Creactive protein. PTX3 binds to various microorganisms and has protective effects against sepsis in animal models. In human studies, high blood levels of PTX3 are associated with sepsis severity, suggesting a potential use as a biomarker. Furthermore, Tipoe et al. performed a systematic review and meta-analysis of plasminogen activator inhibitor-1 (PAI-1) in sepsis. PAI-1 level is increased in patients, and may be used as a predictor of disease severity and all-cause mortality in sepsis.

Low expression levels of human leukocyte antigen D related (HLA-DR) by CD14 leukocytes are increasingly used as a marker of sepsis-induced immunosuppression. Tamulyte et al. report on a prospective cohort assessing monocyte HLA-DR by a pointof-care test. They demonstrate the principle feasibility of the approach, which may help to identify patients with a higher risk of worse outcomes due to sepsis. Excitingly, the development of novel assays for immune profiling patients with severe infections has made huge progress and is anticipated to enable personalized medicine trials in the very near future.

The most widely studied immunomodulatory drugs in sepsis are corticosteroids. A large number of high quality RCTs in critically ill adults are available, as nicely reviewed by Heming et al. The immunomodulatory effects of statins have been recognized for some time. Braga Filho et al. used a mouse model of CLP to demonstrate the positive impact on simvastatin on survival and immune modulation.

## CONCLUSIONS

In summary, the knowledge on the pathophysiology of sepsis has expanded in recent years. Many original and review article in this Research Topic describe experimental and clinical studies pointing to new candidates for biomarkers and interventions, which may ultimately pave the way toward personalized care for sepsis. The availability of improved immunophenotyping approaches, coupled with a number of candidate drugs currently under investigation is promising. The marked heterogeneity of sepsis advocates for highly selective interventions, targeted at populations selected for patients who are more likely to respond to, and to benefit from therapeutic interventions. The success of this endeavor will depend on the capability to incorporate effective strategies for patient selection and targeted treatment allocation. As a promising sign, a number of consortia have recently been launched to advance personalized immunotherapy in sepsis. As nicely discussed by Talisa et al. trials using adaptive enrichment and response adaptive randomization may be best suited to tackle the challenges of developing effective treatments for clinical practice. Recently, large international randomized, embedded, multifactorial adaptive platform trials (originally developed in the field of cancer clinical research) have been launched in infectious diseases and hopefully will boost the efficiency and impact of sepsis research. These advances may motivate further personalized medicine trials leading to sustainable outcome improvements for this major killer disease.

### AUTHOR CONTRIBUTIONS

All authors contributed to the article and approved the submitted version.

### FUNDING

LS was supported by a Practitioner Fellowship of the National Health and Medical Research Council of Australia and New Zealand, and by the Children's Hospital Foundation, Brisbane, Australia. JT was supported by the Swiss National Science Foundation (PZ00P3\_161147, PZ00P3\_183777) and several Swiss foundations (Bangerter-Rhyner, Palatin, Olga Mayenfisch). TR was supported by the Swiss National Science

### REFERENCES

Foundation (173123), the Horizon 2020 Marie Skłodowska-Curie Action Innovative Training Network European Sepsis Academy (676129), and by the Horizon 2020 grant ImmunoSep (847422).

### ACKNOWLEDGMENTS

We wish to convey our appreciation to all the authors who have participated in this Research Topic and the reviewers for their insightful comments.

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Schlapbach, Trück and Roger. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Yin and Yang of Tyrosine Kinase inhibition During experimental Polymicrobial sepsis

*Cassiano Felippe Gonçalves-de-Albuquerque1,2,3‡, Ina Rohwedder2‡, Adriana Ribeiro Silva1 , Alessandra Silveira Ferreira1 , Angela R. M. Kurz1,2†, Céline Cougoule4 , Sarah Klapproth2 , Tanja Eggersmann2 , Johnatas D. Silva5 , Gisele Pena de Oliveira5 , Vera Luiza Capelozzi6 , Gabriel Gutfilen Schlesinger 1 , Edlaine Rijo Costa7 , Rita de Cassia Elias Estrela Marins4,8, Attila Mócsai9 , Isabelle Maridonneau-Parini4 , Barbara Walzog2 , Patricia Rieken Macedo Rocco5 , Markus Sperandio2‡ and Hugo Caire de Castro-Faria-Neto1 \*‡*

*1 Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil, 2Walter Brendel Centre, Department of Cardiovascular Physiology and Pathophysiology, Klinikum der Universität, Ludwig Maximilians University München, Munich, Germany, 3 Laboratório de Imunofarmacologia, Instituto Biomédico, Universidade Federal do Estado do Rio de Janeiro, Rio de Janeiro, Brazil, 4 Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, Toulouse, France, 5 Laboratory of Pulmonary Investigation, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, 6Laboratório de Genômica Pulmonar, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil, 7 Laboratorio de Farmacologia, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, 8 Laboratório de Pesquisa Clínica em DST e AIDS, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil, 9MTA-SE "Lendület" Inflammation Physiology Research Group, Department of Physiology, Semmelweis University, Budapest, Hungary*

Neutrophils are the first cells of our immune system to arrive at the site of inflammation. They release cytokines, e.g., chemokines, to attract further immune cells, but also actively start to phagocytose and kill pathogens. In the case of sepsis, this tightly regulated host defense mechanism can become uncontrolled and hyperactive resulting in severe organ damage. Currently, no effective therapy is available to fight sepsis; therefore, novel treatment targets that could prevent excessive inflammatory responses are warranted. Src Family tyrosine Kinases (SFK), a group of tyrosine kinases, have been shown to play a major role in regulating immune cell recruitment and host defense. Leukocytes with SFK depletion display severe spreading and migration defects along with reduced cytokine production. Thus, we investigated the effects of dasatinib, a tyrosine kinase inhibitor, with a strong inhibitory capacity on SFKs during sterile inflammation and polymicrobial sepsis in mice. We found that dasatinib-treated mice displayed diminished leukocyte adhesion and extravasation in tumor necrosis factor-α-stimulated cremaster muscle venules *in vivo*. In polymicrobial sepsis, sepsis severity, organ damage, and clinical outcome improved in a dose-dependent fashion pointing toward an optimal therapeutic window for dasatinib dosage during polymicrobial sepsis. Dasatinib treatment may, therefore, provide a balanced immune response by preventing an overshooting inflammatory reaction on the one side and bacterial overgrowth on the other side.

Keywords: sepsis, inflammation, dasatinib, Src tyrosine kinase, leukocyte trafficking

#### *Edited by:*

*Thierry Roger, Centre Hospitalier Universitaire Vaudois (CHUV), Switzerland*

#### *Reviewed by:*

*Sang Hoon Rhee, Oakland University, United States Marisa Mariel Fernandez, Instituto de Estudios de la Inmunidad Humoral (IDEHU), Argentina*

#### *\*Correspondence:*

*Hugo Caire de Castro-Faria-Neto<EMAIL_ADDRESS>
#### *† Present address:*

*Angela R. M. Kurz, The Centenary Institute, Newtown, NSW, Australia*

*‡ These authors have contributed equally to this work.*

#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 24 January 2018 Accepted: 11 April 2018 Published: 30 April 2018*

#### *Citation:*

*Gonçalves-de-Albuquerque CF, Rohwedder I, Silva AR, Ferreira AS, Kurz ARM, Cougoule C, Klapproth S, Eggersmann T, Silva JD, Oliveira GPd, Capelozzi VL, Schlesinger GG, Costa ER, Estrela Marins RdCE, Mócsai A, Maridonneau-Parini I, Walzog B, Rocco PRM, Sperandio M and Castro-Faria-Neto HCd (2018) The Yin and Yang of Tyrosine Kinase Inhibition During Experimental Polymicrobial Sepsis. Front. Immunol. 9:901. doi: 10.3389/fimmu.2018.00901*

**10**

### INTRODUCTION

Sepsis is a life-threatening systemic inflammatory condition which results in shock, multiple organ dysfunction, and eventually death (1, 2). It is characterized by a cytokine storm released from myeloid cells during an inadequate antimicrobial response to invading pathogens (3).  The global incidence has been estimated to be 31 million including 6 million fatalities (5).

Neutrophil activation and invasion into inflamed tissue is a critical step in the host's fight against an infection. Under normal circumstances, extravasation of neutrophils is tightly regulated by different receptors and ligands on both the endothelium and neutrophils (6). In order to transmigrate, neutrophils need to roll, adhere, and crawl along the activated endothelium to find a spot for extravasation. Following extravasation, neutrophils release cytokines like interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α to attract more immune cells (7–9). In addition, they start to phagocytose pathogens. In sepsis, pathophysiologic processes are rather caused through an exuberant host response by immune cells against the invading microorganisms than through the direct effects of microbes itself (10). In this respect, a balanced immune response depends on regulatory mechanisms modulating the intensity of the immune response. The Src-family of tyrosine kinases (SFKs) are a group of signaling enzymes with diverse biological effects including, but not limited to, cell proliferation, survival, migration, and metastasis (11–13). SFKs are the largest family of cytoplasmic tyrosine kinases expressed in innate immune cells. The presence of those may vary between innate immune cells, with Hck, Fgr, and Lyn being the most prominently expressed SFKs in monocytes, macrophages, granulocytes, and dendritic cells (14). SFKs bind directly to the cytoplasmatic tail of activated integrins and are responsible for the majority of protein phosphorylations involved in integrin outside-in signaling. Various studies using knockout mice or inhibitors demonstrated the importance of SFKs in host defense and inflammation (15–19), including adhesion and transmigration during leukocyte recruitment (20). Because of these findings, tyrosine kinase inhibitors, originally designed for cancer therapy, have been studied for their role as immune-modulating drugs. Dasatinib, a multi-kinase inhibitor with strong effects on SFKs, acts on both Abl- and Src-family tyrosine kinases (21), and is currently used in patients with chronic myeloid leukemia and acute lymphoblastic leukemia with Philadelphia positive chromosome (Ph+) (22, 23). Besides its effect on malignant cells, dasatinib decreases systemic TNF-α production after LPS injection in a Src and Bruton's tyrosine kinase dependent fashion (24) and reduced lung injury in a dose-dependent manner (25). Additionally, dasatinib treatment reduced chemokine secretion by neutrophils and bone marrow-derived macrophages, suggesting that SFKs are also critical regulators of chemokine secretion in myeloid cells (26). As immune responses to pathogens prevent their dissemination and favor their elimination by the host, there is a concomitant risk of exaggerated immune responses, which may lead to tissue and organ damage. Thus, we hypothesized that immunomodulatory drugs balancing immune responses may be beneficial during systemic severe infection. To test this, we investigated the safety and efficacy of the tyrosine kinase inhibitor dasatinib during inflammation and sepsis. We show that dasatinib diminished the recruitment of leukocytes to the site of inflammation in the inflamed cremaster muscle model. In addition, in a model of polymicrobial sepsis, dasatinib treatment improved survival and sepsis severity in mice and reduced organ damage in a dose-dependent manner with an optimal dose for survival.

### MATERIALS AND METHODS

### Animals

We used male Swiss Webster (SW) mice (25–30 g) from the Oswaldo Cruz Foundation breeding unit, Rio de Janeiro, Brazil. Animals were lodged at 22°C with a 12-h light/dark cycle and free access to food and water. For *in vivo* cremaster muscle experiments *Lyz2*GFP and *Hck−/−Fgr−/−Lyn−/−* (SFK-ko) mice on a C57Bl/6 background were used (27–29). These mice were maintained at the Walter Brendel Center for Experimental Medicine, Ludwig Maximilians Universität, Munich, Germany and accommodated in a barrier facility under SPF conditions. Mice used in the experiment were at least 8 weeks of age and of healthy appearance.

### Pharmacokinetic Analysis

Pharmacokinetic evaluations were performed after the second administration of dasatinib (1 mg/kg). The administrations were made at the following time points: 30 min before CLP and 6 h after CLP. Blood samples were drawn at 0.25, 0.5, 0.75, 1, 2, 4, 8, 16, and 23.75 h (**Figure 1C**).

Dasatinib levels in plasma were determined using a validated high-performance liquid chromatography–tandem mass spectrometry method (HPLC–MS/MS). HPLC system (1200 series, Agilent Technologies, Germany) is connected with API 3200 triple quadrupole mass spectrometer (SCIEX, Toronto, ON, Canada) using multiple reaction monitoring (MRM). The MRM transitions monitored were *m/z* 488.2 → 401.3 for dasatinib, *m/z* 629.4 → 155.2 for internal standard.

### 3D Chemotaxis Assay

The analysis of migration in collagen gels was performed in μ-slide chemotaxis chambers (IBIDI, Planegg, Germany). A gel–cell mixture consisting of 3 × 105 neutrophils in 1.5 mg/mL type I rat tail collagen (IBIDI) was applied to the middle channel of the 3D chamber and left at 37°C for 5 min for gelation. After application of 100 nM fMLP for 20 min at 37°C, time-lapse videos were recorded for 10 min using an Axiovert 200 M microscope (Zeiss, Jena, Germany) equipped with a Plan-Apochromat 10×/0.75NA objective, AxioCam HR digital camera, and a temperaturecontrolled environmental chamber. Migration tracks were analyzed offline with the Image J software. Single cell migration tracks and rose plots were generated using the IBIDI Chemotaxis software.

### Intravital Microscopy

We applied intravital microscopy in exteriorized inflamed cremaster muscle venules of *Lyz2* GFP and SFK-ko mice, as

described (30). Briefly, mice were treated with intrascrotal injection of 500 ng TNF-α, 2 h prior to microscopy. Mice were then anesthetized with intraperitoneal (i.p.) injection of ketamine (125 mg/kg body weight, Ketalar; Parke-Davis, Morris Plains, NJ, USA), and xylazine (12.5 mg/kg body weight; Phoenix Scientific, Inc., St. Joseph, MO, USA). Thereafter, mice were placed on a heating pad to maintain body temperature, intubated, and the left carotid artery cannulated for blood sampling and systemic antibody administration. To maintain a neutral fluid balance, mice were given heparinized saline 0.2 mL/h i.v. throughout the experiment. Intravital microscopy was conducted on an upright conventional fluorescence microscope (Olympus BX51WI, Tokio, Japan) with a saline immersion objective (SW40/0.75 numerical aperture, Zeiss, Jena, Germany).

### Cremaster Muscle Preparation

The surgical preparation of the cremaster muscle was conducted as described (31). Shortly, after surgically opening the scrotum, the cremaster muscle was exteriorized and spread over a cover glass. The epididymis and testis were gently pinned aside giving full microscopic access to the cremaster muscle microcirculation. Experiments were recorded *via* a CCD camera system (CF8/1, Kappa, Gleichen, Germany) on a Panasonic S-VHS recorder and on hard-drive using virtual dub software. The cremaster muscle was superfused with thermocontrolled (35°C) bicarbonate-buffered saline. Postcapillary venules under observation ranged from 25 to 45 µm in diameter. Blood samples were taken during and after the experiment and WBC/neutrophil counts determined using ProCyte Dx Hematology Analyzer (IDEXX, Westbrook, ME, USA). Venular diameter, venular vessel segment length, and leukocyte rolling velocity were assessed using Fiji software (32). Venular centerline red blood cell velocity in the cremaster muscle preparation was measured during the experiment using a dual photodiode and a digital online cross-correlation program (Circusoft Instrumentation, Hockessin, DE, USA).

In a second set of experiments, the number of transmigrated cells was determined. For this approach, mice were treated as described above. After exteriorization, mouse cremaster muscles were dissected and fixed by 4% PFA (AppliChem GmbH, Darmstadt, Germany). Thereafter, cremaster muscle whole mounts were stained using Giemsa (Merck Millipore, Darmstadt, Germany) and the number of transmigrated cells/mm2 assessed using a Zeiss Axioskop 40 microscope with an oil immersion objective 100×, 1.25NA (Zeiss, Jena, Germany). Micrographic images are shown using an oil immersion objective 40×, 1.3NA (Zeiss).

### Dasatinib Treatment

*Lyz2GFP mice* received dasatinib (1, 10, or 20 mg/kg) by gavage in a volume of 100 μL/10 g methylcellulose. Control mice received methylcellulose alone. *Swiss* male mice received dasatinib (1 or 10 mg/kg) by gavage in a volume of 100 µL per animal 30 min before, 6 and 24 h after the induction of sepsis. Control animals received DMSO/saline solution (vehicle) same volume of dasatinb treatment. We based our treatment on dasatinib pharmacokinetics data reported by Ref. (33, 34). In acute experiments with end point at 24 h after cecal ligation and puncture (CLP) the animals received only the two first doses of the drugs (**Figures 1A–C**).

### CLP Model

Swiss mice were anesthetized by intraperitoneal injections of ketamine (100 mg/kg, Cristália) and xylazine (10 mg/kg, Syntec) 10–15 min prior to surgery. The cecal ligation was done below the ileocecal valve and the cecum was perforated four times with an 18G needle. A small amount of fecal material was squeezed from the holes before reinsertion of the cecum in the abdominal cavity. Volemic reposition was made with 1 mL of sterile saline subcutaneously. The animals received meropenem (10 mg/kg) diluted in salina with glucose at 20% intraperitoneally at 6, 24, and 48 h after CLP in 500 µL volume. Sham-operated animals constituted the control and received the same volume reposition and antibiotic treatment administered to CLP animals. After 24 h the animals were submitted to euthanasia using isoflurane (Cristália), and the peritoneal cavity was washed with PBS for colony-forming unit (CFU) analysis and total and differential leukocyte counting (**Figures 1A,B**).

### Biochemical Analysis

Mice were kept in a 12-h fasting with water *ad libitum*, and then blood was collected by cardiac puncture. Serum was separated by centrifugation and used for the quantification of albumin, creatinine, alanine, aspartate aminotransaminase. The quantifications were made using the dry chemistry methodology (Ortho Clinical—Johnson & Johnson) for biochemical parameters.

### Assessment of Sepsis Severity

At 24 h after CLP, mice were scored for severity of sepsis. In this assessment, higher scores reflect increased severity. Mice were scored based on the following variables: piloerection, curved trunk, alterations in gait, seizures, lethargy, respiratory rate, lacrimation, grip strength, feces alterations, body tone, and body temperature alterations [adapted from Ref. (35, 36)]. Each animal received a total score between 1 and 11 and was ranked as: 1–3 (mild sepsis); 4–7 (moderate sepsis); and 8–11 (severe sepsis)*.* In our experimental conditions, most animals were ranked as moderate sepsis.

### Peritoneal Lavage

Briefly, mice were submitted to euthanasia 24 h after surgery using isoflurane (Cristália). The peritoneal cavity was washed with 3 mL of cold sterile saline in the laminar flow cabinet. The peritoneal washes were plated in Difco tryptic soy agar (TSA) (BD) for further analysis of bacterial growth through the count of CFU.

The peritoneal washes were also used for total cell count. Red blood cells were lysed using Turk solution (2% acetic acid) and total cell count was carried out using Neubauer chamber (Neubauer Improved). Differential leukocyte count was performed in cytocentrifuged smears stained with panotic (Laborclin). The supernatant was collected by centrifugation and stored at −20°C for further cytokine quantification.

### Cytokine and LTB4 Measurement

Tumor necrosis factor-α, IL-10, and IL-1β from the supernatant of peritoneal fluid or plasma were measured by enzyme-linked immunoabsorbant assay (ELISA, Duo set kit—R&D systems, Minneapolis, MN, USA) according to the manufacturer's instruction. LTB4 was measured by enzyme immunoassay (EIA, Ann Arbor, MI, USA) according to the manufacturer's instruction.

### CFU Counts

The number of CFU was determined in peritoneal lavage fluid, blood, and other organs that were diluted 1:10,000 and 1:1,000 and incubated under aerobic and sterile conditions on Difco TSA for 24 h at 37°C. The number of bacterial colonies were counted and expressed as CFU/mL.

### Histology

Histological analysis of omentum was performed as previously described (37, 38). Briefly, omentums were collected, fixed in 5% buffered formaldehyde and paraffin-embedded. Tissue sections (4 µm thick) were stained with hematoxyline and eosin for histomorphological analysis.

### Kidney, Small Intestine, and Liver Tissue Damage

The left kidney and the distal part of the right lobe of the liver were also removed after euthanasia. The tissues were fixed in 5% buffered formaldehyde, paraffin-embedded, and sections (4-µm thick) obtained. Liver sections were stained with hematoxylin–eosin, whereas kidney tissue was stained with periodic acid–Schiff reagent to visualize the basement membrane. 10 to 15 fields per section from random tubular regions of the renal cortex and liver parenchyma were captured at a magnification of 400×. Renal tubular damage was defined as tubular epithelial swelling, loss of brush border, vacuolar degeneration, and desquamation. A five-point, semi-quantitative, severity-based scoring system was used to assess each lesion parameter, graded as: 0 = normal tissue; 1 = 1–25%; 2 = 26–50%; 3 = 51–75%; and 4 = 76–100% of examined tissue.

In liver tissue, 10 fields per liver zone (central, lobular, and portal) were captured at a magnification of 400×. The ratio between sinusoidal cells and total cells was computed and expressed as percentage.

Image-Pro Plus 6.3 for Windows (Media Cybernetics, Silver Spring, MD, USA) was used for all analyses.

### DNA Measurement

Extracellular DNA was measured as an indicative of neutrophil extracellular trap (NET) formation. The DNA was quantified in the free cell peritoneal lavage fluid by using the Picogreen dsDNA kit (Invitrogen) according to the manufacturer's instructions.

### DNAse Treatment

In some experiments, we also treated CLP or CLP+ dasatinib animals with DNase (5 mg/kg dissolved in saline solution enriched with 2 mM CaCl2, i.p., 1 h after CLP). The CFU was analyzed in peritoneal lavage at an earlier time point (3 h after CLP).

### Plasma Non-Esterified Fatty Acid (NEFA) Quantification

Plasma concentrations of the predominant NEFA—palmitic, oleic, linoleic, palmitoleic, and stearic acids—were assessed by a colorimetric assay (Zen-Bio, Inc.) (39).

### Phagocytosis Assay

Whole blood was collected from mice receiving dasatinib or vehicle. The assay was performed using the pHrodo *E. coli* bioparticles phagocytosis kit for flow cytometry (Invitrogen) according to the manufacturer's instructions.

### Statistical Analysis

Data are represented as mean ± SEM and statistically analyzed by analysis of variance (one-way ANOVA) followed by Tukey and Student's *t*-test. Survival curves and comparisons between curves were assessed using the Mantel–Cox log-rank test. \**P* values < 0.05 and \*\*\**P* values < 0.001 were considered significant.

### RESULTS

### Dasatinib Treatment Increased Rolling Velocity and Severely Impaired Neutrophil Adhesion *In Vivo*

Dasatinib, a potent Src family kinase inhibitor is known to modulate immune responses (40). Therefore, we set out to evaluate the effect of dasatinib on leukocyte recruitment in an *in vivo* model of TNF-α (2 h) induced inflammation of the mouse cremaster muscle using *Lyz2*GFP mice. Dasatinib (2, 10, or 20 mg/kg) was given orally 3 h prior to the exteriorization of the cremaster muscle. Observation of leukocyte rolling in cremaster muscle venules revealed a significant increase in the number of rolling leukocytes in the presence of dasatinib (**Figure 2A**). Because dasatinib is a broad-spectrum tyrosine kinase inhibitor, we additionally performed analysis of leukocyte recruitment in hck−/− fgr−/− lyn−/− (SFK-ko) animals. In this model, all neutrophil-specific SFKs are deleted and, therefore, display a positive control for dasatinib specificity. We obtained comparable numbers of rolling cells/min in SFK-ko animals after TNF-α stimulation to 10 and 20 mg/kg dasatinib administration (Figure S1A in Supplementary Material). Because absolute numbers of rolling cells are influenced by changes in WBC count, we determined systemic leukocyte counts for each experiment and detected a dose-dependent increase in WBC counts following dasatinib application (Figure S1B in Supplementary Material). SFK-ko animals also showed an increase in the WBC count, following TNF-α stimulation, indicating that this is an SFK-dependent mechanism. We then assessed leukocyte rolling flux fraction, which is defined by the number of rolling leukocytes/min divided by the WBC count. Interestingly, this normalization reduced the observed increase in rolling (**Figure 2B**), indicating that dasatinib did not alter the relative number of rolling cells, but increased total circulating leukocytes. Next, we analyzed leukocyte rolling velocities and found a significant and dose-dependent increase in rolling velocity in the presence of dasatinib and in the SFK-ko mice (2 mg/kg 10.1 µm/s and 20 mg/kg 15.0 µm/s vs. 6.9 µm/s in WT control mice; **Figure 2C**; Figure S1C in Supplementary Material). This suggests that dasatinib inhibits Src kinase dependent intermediate activation of beta2 integrins, a process known to modulate rolling velocities in inflamed tissues (41). Interestingly, absolute number of adherent leukocytes in TNF-α (2 h) stimulated cremaster muscle venules of dasatinib-pretreated mice showed only minor changes (**Figure 2D**). However, after normalizing the number of adherent cells to changes in WBC revealed a severe and dosedependent leukocyte adhesion defect (**Figure 2E**). This decrease was also visible in SFK-ko animals (Figure S1D in Supplementary Material). To exclude that these effects were due to changes in surface expression of rolling- and adhesion-relevant molecules, we performed FACS analysis of leukocyte surface molecules (Figure S1E in Supplementary Material). No major differences in surface expression could be detected for CD18, CD11a, CD11b, CD62L, PSGL1, CXCR2, and CD44.

Overall, these findings demonstrate that SFK inhibition by dasatinib significantly increases leukocyte rolling velocity and reduces leukocyte adhesion in inflamed postcapillary venules *in vivo*.

### Dasatinib Treatment Strongly Reduced Leukocyte Extravasation

To extravasate into inflamed tissue, leukocytes need to crawl along the endothelial wall to find an appropriate spot for extravasation. We performed time-lapse fluorescence video microscopy in TNF-α (2 h) stimulated cremaster muscle venules and tracked GFP-fluorescent crawling leukocytes in control or dasatinib (10 mg/kg)-treated Lyz2GFP mice. In contrast to previous observations of neutrophil 2D migration in Zigmond chambers (42), dasatinib had almost no detectable effect on 2D neutrophil crawling *in vivo* (**Figure 3A**). No significant differences in crawling velocity or Euclidean distance were observed (**Figure 3B**), indicating that those cells which adhere in the presence of dasatinib display no further migration defect.

The crossing of the vascular wall is the last step in the leukocyte adhesion cascade. To quantify extravasation, we performed Giemsa staining of fixed cremaster muscle tissues of control and dasatinib (10 mg/kg) treated mice after TNF-α stimulation and counted perivascular leukocytes (**Figure 3C**). Quantification of extravasated cells revealed a significant inhibition of leukocyte extravasation by dasatinib compared to control animals (394 vs. 632 cells/mm2 , **Figure 3D**). Likewise in SFK-ko mice, the number of extravasated cells was decreased to a similar degree (430 cells/mm2 ). This further strengthens our hypothesis of dasatinib acting mostly on SFKs during leukocyte recruitment. A more detailed analysis of leukocyte subtypes crossing the vessel wall showed that dasatinib mainly inhibited neutrophil extravasation (**Figure 3E**). In contrast to intraluminal crawling, leukocyte migration in the interstitium occurs in a 3D environment and is integrin-independent and eventually also SFK independent. We, therefore, performed *in vitro* 3D migration experiments to investigate the effects of dasatinib on integrin-independent migration. Indeed, analyzing migration of isolated leukocytes

in a 3D collagen gel matrix revealed no alteration of leukocyte migration behavior in the presence of dasatinib (**Figure 3F**; Figure S1F in Supplementary Material). The Euclidean distance as well as their migration velocity was unchanged. This indicates that SFKs, similar to leukocyte integrins (43), are dispensable for interstitial migration once leukocytes managed to overcome the vascular barrier.

### Dasatinib Dose-Dependent Effect on Survival and Severity of Sepsis After CLP

Our *in vitro* and *in vivo* findings described above suggest a potential role of dasatinib treatment on the outcome of sepsis. We, therefore, tested dasatinib administration (1 and 10 mg/kg) in the CLP model of induced sepsis. Our first step was to evaluate the bioavailability of dasatinib by measuring its concentration in the plasma of septic animals. For that, we performed pharmacokinetics analyses and compared the plasma concentration of 1 mg/kg dasatinib after administration to sham or CLP animals (**Figure 4A**). Septic animals had lower peak values of the drug than treated sham animals but their plasma levels remained at pharmacological levels up to 24 h after administration. Of note, plasma concentrations in both animals remained markedly above the concentration (14.9 ng/mL) able to inhibit 90% of phosphorylation of pBCR-ABL protein (34). We next induced polymicrobial sepsis using the CLP model to test for survival and clinical scores in septic mice. Sham-treated animal showed 100% survival rate after 7 days. Following CLP, we observed a survival rate of 50% after 7 days in control animals, with the highest mortality observed between day 1 and 2 after CLP. In contrast, administration of dasatinib at 1 mg/kg protected the animals from lethal sepsis following CLP (**Figure 4B**). Dasatinib at 1 mg/kg administered 30 min before and 6 and 24 h after CLP resulted in an 80% survival rate 7 days after CLP. Interestingly, a higher dose (10 mg/kg) of dasatinib had an opposite effect, with a mortality rate increasing to 85%.

The beneficial effect of low dasatinib doses was also detected in the severity score. As shown in **Figure 4C**, 1 mg/kg dasatinib improved severity scores compared to untreated CLP animals and resulted in only moderate sepsis scores. Again 10 mg/kg dasatinib reversed this effect. Sham-treated animals did not present any sign of disease.

### Low Dosage of Dasatinib Decreased Organ Dysfunction in Septic Animals

To evaluate the protective effect of dasatinib treatment in septic animals in more detail, we analyzed the impact of dasatinib

Figure 3 | Dasatinib treatment strongly reduces leukocyte extravasation. *In vivo* crawling experiments were analyzed in rmTNF-α-stimulated Cremaster muscle tissue of Lyz2 GFP mice, pretreated orally with 10 mg/kg dasatinib in Methylcellulose, or with Methylcellulose alone (control). Time-lapse movies over 15 min were performed and leukocytes visualized by their GFP signal. Extravasated leukocytes were analyzed with Giemsa staining in rmTNF-α-stimulated fixed cremaster muscle tissue of Lyz2 GFP mice, treated orally with 10 mg/kg dasatinib in methylcellulose, or with methylcellulose alone. Data are presented as mean ± SEM. (A) Representative single cell migration tracks and rose plots for intraluminal crawling are displayed. Red lines indicate migration in, black lines migration against flow direction. At least 80 cells were analyzed for each strain (B) Evaluation of crawling velocity and Euclidian distance of crawling cells (C) Representative images of cremaster muscle whole mounts after Giemsa staining. Scale bar: 10 µm (D) Total number of extravasated cells/mm2 in muscle tissue in close proximity to a vessel. Star indicates significance over control. (E) Differential total cell counts of polymorphonuclear cells, eosinophils, and other cells. (F) Evaluation of migration velocity and Euclidian distance of crawling PMNs with or without dasatinib (\**P* < 0.05). Statistical analysis: Student's *t*-test.

treatment in sepsis-induced organ dysfunction. We measured plasma biological markers for kidney (creatinine) and liver (aspartate and alanine aminotransferase), and lipotoxicity (NEFA). All CLP animals displayed significantly increased levels of creatinine, aspartate aminotransferase, NEFA, indicating severe organ damage caused by CLP induced sepsis (**Figures 5A–D**).

Figure 4 | Dose-dependent effect of dasatinib on survival and severity of sepsis after cecal ligation and puncture (CLP). *Swiss* mice were submitted to CLP. Sham-treated animals were used as control. Data are presented as mean ± SEM. (A) Dasatinib was given orally 30 min before and 6 and 24 h after CLP. Dasatinib concentration of blood samples taken at indicated timepoints is displayed. (B) The survival rate was quantified for 7 days (144 h) in sham mice, untreated animals or each treated with dasatinib at 1 or 10 mg/kg dosage. (C) Clinical score was assessed 24 h after CLP. Each dot represents one animal. 1–3 points in the clinical score corresponds to a mild sepsis, 4–7 points corresponds to a moderate sepsis, and 8–11 points corresponds to severe sepsis. The animals were treated with dasatinib 1 or 10 mg/kg orally 30 min before, 6 and 24 h after CLP procedure. At least two independent experiments were performed. Statistical analysis: one-way ANOVA followed by Tukey \**P* < 0.05, \*\**P* < 0.01, and \*\*\**P* < 0.001, for Figure 3B and Mantel–Cox log-rank test \**P* values < 0.05, for Figure 3C. The number of animals per group range from 3 to 4 for pharmacokinetics, 7 to 31 for clinical score, and 10 to 11 per group from mortality.

Dasatinib treatment at 1 mg/kg lowered these levels in CLP mice indicating that dasatinib can partly rescue sepsis-induced organ damage. Reduced plasma levels of albumin and glucose after CLP could not be rescued by dasatinib (Figures S2A,B in Supplementary Material).

In agreement with systemic biochemical markers of organ dysfunction, histological alterations were detected in the liver (first column), the small intestine (second column), and the kidney (third column) of septic mice, compared to control group (**Figure 5E**). CLP induced liver and kidney steatosis and edema in small intestine villi. However, treatment with 1 mg/kg dasatinib prevented these alterations. Histological changes were scored from 0 (without alterations) to 4 (more extensive lesions) and we could detect an overall decrease in the severity of the organ damage in CLP mice treated with dasatinib to the score levels of CLP mice (**Figure 5F**). In the omentum, we found some neutrophil infiltration due to the inflammatory reaction induced by surgery in sham animals. Dasatinib treatment did not affect omentum morphology or leukocyte infiltration in sham animals (Figure S2C in Supplementary Material). In septic mice, the omentum seems to be liquefied due to the intensity of the acute inflammatory response taking place in the peritoneal cavity. In dasatinib-treated animals, the omentum histology was similar to the sham conditions, reinforcing the protective role of dasatinib in CLP induced sepsis.

### Dasatinib Treatment Impaired the Number of Leukocytes in the Peritoneal Cavity and Decreased Concentration of Inflammatory Mediators

We next investigated the effect of dasatinib treatment in leukocyte accumulation and inflammatory mediators in more detail. For this purpose, we analyzed cell accumulation in the peritoneal cavity 24 h after CLP (**Figures 6A–C**). As expected, septic animals had higher numbers of mononuclear cells and neutrophils in the inflamed peritoneal cavity as compared to sham-treated animals (**Figures 6A–C**). Treatment with dasatinib (1 mg/kg) significantly lowered both mononuclear cell and neutrophil accumulation in the peritoneal cavity (**Figures 6B,C**). This finding is in accordance to our previous data of reduced leukocyte extravasation in inflamed cremaster muscle tissue after dasatinib treatment.

Additionally, we analyzed the effect of dasatinib (1 mg/kg) on cytokine, and chemokine production. We measured plasma levels of TNF-α, IL-6, and IL-10 in dasatinib-treated animals 24 h after CLP and compared them to sham-treated mice. Septic animals presented elevated levels of all measured cytokines (**Figures 6D–F**). Animals treated with dasatinib at 1 mg/kg dose presented significantly lower levels of all cytokines indicating a reduced extend of inflammation. We also measured the levels of cytokines, chemokines, and lipid mediators in the peritoneal lavage of septic mice. The levels of TNF-α and IL-6 were increased in septic mice and treatment with dasatinib decreased their levels (**Figures 6G,H**). Dasatinib administration did not affect IL-1β levels (**Figure 6I**). Septic mice also displayed increased peritoneal levels of LTB4 and CXCL1/KC. Likewise 1 mg/kg dasatinib decreased LTB4 and CXCL1/KC levels (**Figures 6J,K**), while MCP1 levels remained at the same levels as in the CLP group (**Figure 6L**).

### Dasatinib Inhibited Bacterial Growth and Bacterial Spreading in Septic Mice

About 60–70% of patients with sepsis have positive blood cultures, most of them are Gram-negative bacteria (5, 44). In our model, we have a mixed infection with both Gram-negative and Gram-positive bacteria, detected in the peritoneal fluid from septic animals. Interestingly, 1 mg/kg dasatinib significantly reduced CFU counts in the peritoneal fluid (**Figure 7A**). At 10 mg/kg, however, dasatinib-treated CLP animals showed higher CFU numbers compared to CLP (Figure S3A in Supplementary Material). We also evaluated CFU formation in distal organs to assess bacterial translocation and the ability of the organism to fight infection. We could detect high numbers of CFUs in all analyzed organs of CLP animals. Again 1 mg/ kg dasatinib successfully prevented bacterial translocation to the blood (**Figure 7B**), lung, spleen, kidney, and liver (Figures S3B–E in Supplementary Material).

### Dasatinib Treatment Enhanced Neutrophil Functionality

Neutrophils phagocytose microbes, produce ROS, release antimicrobial factors and form NET as part of their arsenal to fight invading organisms (45). In order to explore why treatment with dasatinib was able to decrease CFU numbers despite reducing the numbers of neutrophils at the site of infection, we investigated the effect of dasatinib on the ability of neutrophils to kill bacteria. To evaluate the effect of dasatinib on NET formation, we measured extracellular double-strand DNA *via* fluorimetry in septic animals with and without dasatinib. As shown in Figure S3F in Supplementary Material, the CLP group showed an increase in extracellular DNA content compared to sham animals. Interestingly, treatment with dasatinib did neither alter extracellular DNA levels nor did the disassembling of NETs by DNAse interfere with the ability of dasatinib to decrease CFU numbers (Figure S3G in Supplementary Material).

Next, we checked the production of nitrite as readout for NO production in the peritoneal cavity after treatment with 1 mg/kg dasatinib. NO and superoxide generate antimicrobial molecules called reactive nitrogen species that act together with ROS in damaging cells and microbes (46, 47). Our results show that dasatinib increased local nitrite production during CLP, which contributes to bacterial clearance (**Figure 7C**). Next we examined the effect of dasatinib treatment on neutrophils phagocytosis. Mice were treated with 1 mg/kg dasatinib and neutrophils phagocytosis was determined in whole blood by flow cytometry. Interestingly, compared to untreated animals, animals treated with dasatinib (1 mg/kg) showed increased neutrophil phagocytosis (**Figure 7D**), suggesting a potential enhancing effect on the ability of neutrophil to clear bacteria. Also here we encountered a dose-dependent effect, because treatment with 10 mg/kg dasatinib decreased the ability of neutrophils to phagocytose.

Figure 5 | Low dosage of dasatinib decreases organ dysfunction in septic animals. Swiss mice were submitted cecal ligation and puncture (CLP). Sham-treated animals were used as control. The animals were treated with dasatinib at 1 mg/kg 30 min before and 6 h after CLP. Blood was collected 24 h after CLP procedure, and organs were harvested for HE staining. (A) Creatinine, (B) alanine, (C) aspartate aminotransferase, and (D) non-esterified fatty acid (NEFA) were analyzed. Data are presented as mean ± SEM. (E) Optical microscopy of liver, small intestine, and kidney. In the CLP group, liver hepatocytes around of centrilobular vein and tubular renal cells exhibit apoptosis (arrows) and diffuse vacuolization in the cytoplasm by accumulation of fat (asterisks), thus characterizing liver and kidney steatosis; the small intestine show apoptosis of enterocytes (arrows) and prominent edema of the villi (#). After dasatinib treatment, the integrity of liver hepatocytes, small intestine villi, and tubular renal cells are restored with reduction in apoptosis and steatosis score similar to Ssam and Sham animals treated with dasatinib. Scale bar is 50 µm. (F) Injury score with severity analyses of microscopically visible organ damage. The score ranges from 0 to 4 where 0 means no injury and 4 maximum injuries. Statistical analysis: one-way ANOVA followed by Tukey \**P* < 0.05. The number of animals per group range from 4 to 10.

### DISCUSSION

Sepsis is one of the leading causes of morbidity and mortality in Intensive Care Units, and is associated with increased health-care costs (48, 49). This is complicated by the rise of drug-resistant microorganisms, a growing elderly population, and an increased incidence of immunosuppression (50–54). The failures of antitoll-like receptor 4 antibody, recombinant activated protein C, and anti-TNF-α therapies in clinical trials require a rethinking of sepsis' pathophysiology and therapeutic strategies (8, 55–60). Systematic approaches, such as presented here, could fuel the discovery of promising immunosuppressive or anti-inflammatory drugs that aim at multifunctional targets such as Src family kinases.

Src family kinases play critical roles in a whole variety of pathologies including cancer; in addition, it was shown that Src is involved in inflammation-related signaling pathways (61). Dasatinib is a type I ATP-competitive protein kinase inhibitor (62). SFK-inhibitors affect signaling pathways and downmodulate the inflammatory response. Nevertheless, the current dose of dasatinib (100 mg daily in human) used to treat some leukemia does not induce severe immunosuppression (63). In the present work, we used lower doses of dasatinib (1 and 10 mg per kg). We chose these doses because initial pharmacokinetic experiments showed that dasatinib at 1 mg per kg yields plasma concentrations that remained above the critical concentration to inhibit SFKs.

It is currently accepted that it is not the insult *per se*, but the host's response, that determines severity and outcome in sepsis (4). Therefore, the immunosuppressive action of dasatinib may affect the response against infectious agents and it is expected that high-dose favors the progression of infection with deleterious effects to the host as shown for *Pneumocystis jiroveci* pneumonia (63). On the contrary, lower doses may modulate the immune response affecting and/or preventing tissue damage resulting from host immune response or cellular hyperactivation. In fact, we show here that dasatinib at 10 mg/kg is deleterious to the host fueling infection progression. In contrast, the lower dose of dasatinib (1 mg/kg) showed promising results improving the animal clinical condition and increasing survival. The higher dasatinib dose may inhibit other kinases and proteins impacting on ability of the host to fight the infection effectively because of its potent anti-inflammatory effect. Lower dose of dasatinib decreases the neutrophil migration but does not abrogate it. So the fewer neutrophils that reach the peritoneum remain effective on killing the bacteria and restrain the infection.

Septic patients present systemic inflammation with exacerbated cytokine production, and increased cell migration to the site of infection or sterile inflammation, as shown with CLP mice in this publication. Trafficking of myeloid leukocytes to the site of inflammation is linked to the generation of an appropriate inflammatory environment (20). In this regard, LTB4 is a potent chemotactic agent for neutrophils (64). Its levels increase 24 h after CLP and treatment with dasatinib (1 mg/kg) reduced the levels of LTB4. Dasatinib also decreased the levels of CXCL1/ KC, another potent chemo attractant to neutrophils. CXCL1/ KC is released by resident macrophages and mediates neutrophil accumulation induced by LPS (65).

Src family tyrosine kinases are important components of the signaling pathways initiated by the TLRs (critical for cytokine production) and many cytokines, such as TNF, use Src family kinases in their own signaling pathways (17, 66). In our model, dasatinib reduced the levels of all measured cytokines confirming data from the literature (25, 67), except IL-1β. Impairing TLR4 related signaling pathway inhibits cytokine production including both TNF and IL-10 (68). IL-1β requires the cleavage of the proform (pro-IL-1β) by caspase-1 into its biologically active form (69). However, we cannot exclude that IL-1β detected here may reflect the release of already present IL-1β in the cell which would suggest that the release of IL-1β is independent of Src kinases (70) while the other measured cytokines are upregulated by SFKdependent transcriptional activity, reinforcing the key role of SFK in cytokine production in infectious disease.

Activated neutrophils are able to generate reactive oxygen species, release NETs, increase phagocytosis, and produce nitric oxide (45). We further elucidated the role of tyrosine kinases in these processes and found that dasatinib did not affect NET formation. In contrast, nitric oxide generation was increased in septic mice treated with dasatinib strengthening its potent bactericidal activity at the local level (71, 72).

Phagocytosis is a prime mechanism of bacterial killing. Src-family kinase-deficient leukocytes are less effective than wild-type cells at mediating phagocytosis (73, 74). On the other hand macrophages lacking Src family members Hck, Fgr, and Lyn showed phagocytosis mediated by Fcγ Receptor (75), so phagocytosis obviously can happen independently of neutrophilexpressed SFKs. Interestingly, our experiments revealed that 1 mg/kg of dasatinib increased phagocytotic activity of blood neutrophils compared to control neutrophils, while higher doses (10 mg/kg dasatinib) decreased phagocytosis. The mechanism for this dose-dependent effect of dasatinib on phagocytotic activity in neutrophils is currently unclear and needs further investigations.

Patients with severe sepsis symptoms display metabolic dysfunction with elevated levels of plasma NEFA and lower levels

Figure 6 | Dasatinib treatment results in fewer peritoneal leukocytes along with decreased amounts of inflammatory markers. Swiss mice were submitted cecal ligation and puncture (CLP). Sham-treated animals were used as control. The animals received dasatinib at 1 mg/kg 30 min before and 6 h after CLP. Cells were collected 24 h after CLP procedure by peritoneal lavage to assay total and differential counts. Data are presented as mean ± SEM. (A) Total leukocyte count, (B) mononuclear cell count, and (C) neutrophil cell count are shown. Cytokines were measured by ELISA and LTB4 by EIA. (D–F) Display plasma levels of tumor necrosis factor (TNF)-α (D), interleukin (IL)-6 (E), and IL-10 (F), (G–L) display peritoneal lavage values of TNF-α (G), IL-6 (H), IL-1β (I), LTB4 (J), CXCL1 (K), and MCP1 (L) Statistical analysis: one-way ANOVA followed by Tukey \**P* < 0.05, \*\**P* < 0.01, and \*\*\**P* < 0.001. The number of animals per group range from 6 to 18.

from dasatinib or vehicle treated cells using pHrodo *E coli* bioparticles. Statistical analysis: one-way ANOVA followed by Tukey \**P* < 0.05, \*\**P* < 0.01, and \*\*\**P* < 0.001. The number of animals per group range from 6 to 15.

of albumin (76, 77). NEFA activates TLRs boosting cytokine production (78), induce cell death (79), and inhibit sodium potassium ATPase in several organs, including the lungs (80). In addition, the inhibition of lipogenesis or the increase in lipid oxidation reduces levels of free fatty acids, TNF-α, and IL-6, and reduces liver injury improving survival in sepsis (81, 82). Accordingly, the observed decrease in NEFA levels in the plasma of dasatinib-treated mice might at least in part be responsible for their improved organ function.

Regardless of their clinical potential in septic patients, our study demonstrates that the use of SFK inhibitors needs to be tightly controlled to keep the fine balance between overtreatment with uncontrolled bacterial growth in an immuno-compromised organism and ineffective treatment leading to a hyper-inflammatory response of the host immune system. Keeping this balance at an optimal level will certainly be a challenge and require intensive monitoring. In view of the fact that great efforts have been made to develop tyrosine kinases inhibitors for the therapy

of inflammatory diseases (83), their use might open new doors in modulating the inflammatory response during sepsis and, therefore, improve the outcome of patients suffering from this life-threatening syndrome.

### ETHICS STATEMENT

Animal housing conditions and experimental procedures conformed to institutional regulations and were in accordance with the National Institute of Health guidelines on animal care. The Institutional Animal Welfare Committee approved all procedures described here under license number 002-08, LW36/10, and L15/2015. The animal experiments were approved by the Regierung von Oberbayern, Germany (AZ 55.2-1-54-2531-80- 76/12). Both Institutions follow the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) originally published in 2010 (84).

### AUTHOR CONTRIBUTIONS

Conceptualization: CG-d-A, IR, AS, PR, MS, and HC-F-N. Data curation: CG-d-A, IR, AS, AF, AK, CC, SK, TE, JS, GO, VC, GS, EC, RM, BW, MS, PR, and HC-F-N. Formal analysis: C-G-d-A, IR, AS, AF, AK, and CC. Funding acquisition: HC-F-N, PR, AS, CG-d-A, MS, BW, AM, and IM-P. Investigation: CG-d-A, IR, AS, AF, AK, CC, SK, TE, JS, GS, EC, and RM. Project administration: CG-d-A, IR, AS, CC, PR, BW, PR, MS, and HC-F-N. Supervision: AS, AM, IM-P, PR, MS, and HC-F-N. Validation: CG-d-A, IR, AS, CC, BW, PR, MS, and HC-F-N. Visualization: CA, IR, AS, CC, MS, and HN. Writing—original draft preparation: CG-d-A,

### REFERENCES

IR, AS, MS, and HN. Writing—review and editing: CA, IR, AS, AF, AK, CC, SK, TE, JS, GO, VC, GS, EC, RM, AM, IM-P, BW, PR, MS, PR, and HC-F-N.

### ACKNOWLEDGMENTS

The funders had no role in study design, data collection and analysis, decisions to publish, or preparation of the manuscript. This work was supported by grants from Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Programa Estratégico de Apoio à Pesquisa em Sau´de (PAPES) FIOCRUZ, and Universidade Federal do Estado do Rio de Janeiro (UNIRIO). The authors also acknowledge financial support by the European Community's Seventh Framework Programme (FP7-2007-2013) under grant agreement HEALTH-F4-2011-282095 (TARKINAID to HC-F-N, AM, BW, PR, IMP, MS), and Programa de Produtividade Científica da Universidade Estácio de Sá. In addition, the authors received funding from SFB914 (project A2 to BW and B1 to MS) by the German Research Foundation (DFG) and Else Kroener Fresenius Foundation (2015\_A68 to IR), Bad Homburg v.d.H., Germany. Authors thank Susanne Bierschenk for help with the phagocytosis experiments.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at https://www.frontiersin.org/articles/10.3389/fimmu.2018.00901/ full#supplementary-material.

chronic myeloid leukaemia. *Nat Rev Cancer* (2007) 7(5):345–56. doi:10.1038/ nrc2126

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Gonçalves-de-Albuquerque, Rohwedder, Silva, Ferreira, Kurz, Cougoule, Klapproth, Eggersmann, Silva, Oliveira, Capelozzi, Schlesinger, Costa, Estrela Marins, Mócsai, Maridonneau-Parini, Walzog, Rocco, Sperandio and Castro-Faria-Neto. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# The Surface-Exposed Protein SntA Contributes to Complement Evasion in Zoonotic *Streptococcus suis*

*Simin Deng1 , Tong Xu2 , Qiong Fang1 , Lei Yu1 , Jiaqi Zhu1 , Long Chen1 , Jiahui Liu1 and Rui Zhou1,3,4,5\**

*1State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China, 2College of Life Science and Technology, Huazhong Agriculture University, Wuhan, China, 3Cooperative Innovation Center of Sustainable Pig Production, Wuhan, China, 4 International Research Center for Animal Diseases (MOST), Wuhan, China, 5Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Wuhan, China*

*Streptococcus suis* is an emerging zoonotic pathogen causing streptococcal toxic shock like syndrome (STSLS), meningitis, septicemia, and even sudden death in human and pigs. Serious septicemia indicates this bacterium can evade the host complement surveillance. In our previous study, a functionally unknown protein SntA of *S. suis* has been identified as a heme-binding protein, and contributes to virulence in pigs. SntA can interact with the host antioxidant protein AOP2 and consequently inhibit its antioxidant activity. In the present study, SntA is identified as a cell wall anchored protein that functions as an important player in *S. suis* complement evasion. The C3 deposition and membrane attack complex (MAC) formation on the surface of *sntA*-deleted mutant strain Δ*sntA* are demonstrated to be significantly higher than the parental strain SC-19 and the complementary strain CΔ*sntA*. The abilities of anti-phagocytosis, survival in blood, and *in vivo* colonization of Δ*sntA* are obviously reduced. SntA can interact with C1q and inhibit hemolytic activity *via* the classical pathway. Complement activation assays reveal that SntA can also directly activate classical and lectin pathways, resulting in complement consumption. These two complement evasion strategies may be crucial for the pathogenesis of this zoonotic pathogen. Concerning that SntA is a bifunctional 2′,3′-cyclic nucleotide 2′-phosphodiesterase/3′-nucleotidase in many species of Gram-positive bacteria, these complement evasion strategies may have common biological significance.

Keywords: *Streptococcus suis*, surface protein SntA, C1q, complement evasion, pathogenesis

### INTRODUCTION

*Streptococcus suis* are recognized as an important swine and human pathogen (1). Among the 33 serotypes, *S. suis* serotype 2 (SS2) is the most virulent and prevalent one, which is also an emerging zoonotic pathogen (2). Two large-scale outbreaks of severe human SS2 infection occur in 1998 and 2005 in China causing 229 infections and 52 deaths (3, 4). In 2005, the streptococcal toxic shock like syndrome (STSLS) is first reported to occur in the human. An early burst of inflammatory cytokines could result in the STSLS with death as quickly as 13 h after SS2 infection, and subsequently SS2 breaks through blood–brain barrier (BBB) to cause disease, particularly meningitis (1, 5). Bacterial pathogens evade host innate immune defenses and maintain a high dose in blood causing bacteremia and septicemia. During these processes, the host complement system is an important factor facilitating clearance of bacterial pathogens (6, 7).

The complement system consists of more than 50 plasma and cell surface proteins. As a first line of defense against pathogenic intruders and a mediator between the innate and adaptive immune

#### *Edited by:*

*Thierry Roger, Centre Hospitalier Universitaire Vaudois (CHUV), Switzerland*

#### *Reviewed by:*

*Juan Li, Rockefeller University, United States Eden Ramalho Ferreira, Federal University of São Paulo, Brazil*

> *\*Correspondence: Rui Zhou<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 04 February 2018 Accepted: 27 April 2018 Published: 16 May 2018*

#### *Citation:*

*Deng S, Xu T, Fang Q, Yu L, Zhu J, Chen L, Liu J and Zhou R (2018) The Surface-Exposed Protein SntA Contributes to Complement Evasion in Zoonotic Streptococcus suis. Front. Immunol. 9:1063. doi: 10.3389/fimmu.2018.01063*

response, it plays an essential and efficient role in rapid recognition and elimination for invading pathogens. The complement has three independent but interactive activation pathways: the classical pathway, alternative pathway, and lectin pathway (8). These three different complement pathways are stimulated by different foreign substance through specific recognition molecules (4). All the complement cascades result in the deposition of C3b to amplify the cascades, and mediate phagocytosis and adaptive immune responses by binding to complement receptors; the release of pro-inflammatory anaphylatoxins and chemoattractant C5a and C3a; and formation of membrane attack complex (MAC; C5b-9) then lead to direct lysis of Gram-negative bacteria (9).

Although host complement can rapidly recognized and eliminated foreign microorganisms, it also offers many interference sites that can disrupt this balanced network of protein interactions by complement-binding proteins leading to failure of elimination by host. Complement-binding proteins can be identified from both host and pathogens. These complement evasion mechanisms include (I) recruiting or mimicking of complement regulators; (II) modulating or inhibiting complement by direct interactions; and (III) enzymatic degradation by complement components (4).

C1q is the recognition subunit of C1 complex to trigger the classical complement pathway, following the recognition of IgG or IgM-bearing immune complexes (10). Proteins that interact with C1q have been identified widely in Gram-negative bacteria, such as *Salmonella Minnesota* (11), *Escherichia coli* (12–14), *Klebsiellapneumoniae* (15), *Legionella pneumophila* (16), *Bacillus*  *anthracis* (17), *Moraxella catarrhalis* (12), nontypeable *Haemophilus influenzae* (12), but in Gram-positive bacteria the research is not much, except for Group B *Streptococci* (18, 19), *Streptococcus pyogenes* (20–22) and *Staphylococcus aureus* (23).

In our previous study, surface protein SntA of *S. suis* without any unknown function has been characterized to be a hemebinding protein which involved in the pathogenesis of *S. suis* in pigs. SntA can interact with the host antioxidant protein AOP2 and consequently inhibit its antioxidant activity (24). Complement C1q is identified as another interacting partner of SntA when we screen the SntA binding proteins in the host. In the present study, we demonstrate that SntA is an important player in complement evasion of this important zoonotic pathogen.

### MATERIALS AND METHODS

### Strains and Culture Conditions

*Streptococcus suis* serotype 2 strain SC-19 (GenBank accession number: NZ\_CP020863.1) used in this study was isolated from a sick pig during the epidemic outbreak in Sichuan province of China in 2005. The *S. suis* strains were grown in tryptic soy broth (TSB; Difco, France) or on tryptic soy agar (TSA; Difco) plates supplemented with 5% newborn bovine serum (Sijiqing, Hangzhou, China) at 37°C. The *E. coli* DH5α and BL21 (DE3) strains were grown in LB broth or on LB agar plates at 37°C. The bacterial strains, plasmids, and primers used in this study are listed in **Table 1**.

### Construction of *sntA* Gene Deletion and Complementary Strains

The *sntA*-deleted strain Δ*sntA* was constructed in our previous study (24). To construct *sntA* complementary strain, a DNA fragment containing the entire *sntA* coding sequence and its promoter and terminator was amplified by using primers CΔ*sntA*\_F/CΔ*sntA*\_R. The amplicon was subsequently cloned into *E. coli*-*S. suis* shuttle vector pSET2, resulting in the recombinant plasmid pSET2:*sntA*. This plasmid was transformed into the Δ*sntA* strain, and the resulting complementary strain CΔ*sntA* was screened on TSA agar plates supplemented with 100 µg/ml spectinomycin (Spc). The Spc-resistant colonies were verified by PCR and reverse transcription (RT)-PCR analyses by using three pairs of primers SntA\_F/SntA\_R, 10065\_F/10065\_R, and 10075\_F/10075\_R (**Table 1**).

### Experimental Infection of Mice

To detect the virulence of *sntA* in *S. suis*, a total of 24 female 5-week-old specific-pathogen-free (SPF) Kunming mice (8 mice per group) were intraperitoneally infected with 2 × 109 colony forming unit (CFU)/mouse of SC-19 and Δ*sntA*. Physiological saline was served as a negative control. Then the morbidity, mortality, and clinical symptoms such as limping, swollen joints, shivering, and central nervous system failure of all the mice were observed for 5 days. To evaluate the effect of *sntA* on survival in blood and organs colonization, competitive colonization experiment of SC-19 and Δ*sntA* was performed. 5-week-old SPF Kunming mice (7 mice per group) were co-infected with SC-19 and Δ*sntA* at the ratio 1:1 (5 × 108 CFU/mouse). Bacterial counts in blood, brain, and lung were collected at 6, 12, 24, and 48 h post infection (hpi). Colonization of bacteria in various tissues were analyzed by serial diluted and plated brain and lung samples after homogenizing, and blood samples on TSA agar plates with (Em+) and without (Emc ) 100 µg/ml erythromycin (Em) as described previously (28). The number of live bacteria on TSA agar plates with or without Em was calculated as Δ*sntA*, sum of SC-19 and Δ*sntA*, respectively. The number of SC-19 was calculated as TSA TSA Em Em − + − .

### Bactericidal Assays

In human blood and serum killing assays, experiments to evaluate the survival rate of *S. suis* were carried out as described previously (29). Overnight cultures of the *S. suis* strains in TSB were diluted in 1:100 and grown to mid-log phase without agitation at 37°C. The cultures were diluted to 5 × 107 CFU/ml with physiological saline. 50 µl diluted cultures was mixed with 450 µl fresh human blood for human blood killing assay and 20 µl diluted cultures were mixed with 180 µl of normal human serum (NHS) and complement heat inactivated serum by incubation at 56°C for 30 min for human serum killing assay. The resulting mixtures were incubated at 37°C for 30 min. Live bacteria were counted by plating the serial diluted samples on TSA agar plates. The percentage of live bacteria was subsequently calculated as (CFUafter incubation/CFUin original inoculum) × 100%.

 Experiment to investigate the survival rate of *S. suis* in PMNs was carried out as described previously (6). PMNs were mixed with bacteria at MOI = 1:10 in RPMI-1640 medium (Hyclone, USA) supplemented with 20% freshly non-immune human serum and incubated at 37°C under 5% CO2 for 30 min. The percentage of live bacteria was subsequently calculated as ( ) CFU /CFU 1 % PMN PMN + − × 00 .

In phagocytosis assay, the experiment was carried out by our previous study (30). Briefly, the 1.0 × 106 RAW264.7 cells for each well were pooled into 12-well plates. Then *S. suis* strains in mid-log phase were added to the plates (MOI = 10:1) and incubated for 30 min at 37°C to allow cells to be phagocytized. After incubation, ampicillin was applied to plates for 1 h to kill the extracellular bacteria. Then, the cells were lysed with sterile distilled water on ice and the bacterial CFU were counted on TSA agar plates. These data are presented as means ± SDs from three separate experiments.

### Complement C3 Deposition and MAC Formation Analysis

C3 deposition and MAC formation assays were performed as previous study (31). Overnight cultures of *S. suis* were diluted in 1:100 in TSB and grown to mid-log phase without agitation at 37°C. Bacteria were collected by centrifugation at 6,000 rpm for 5 min. After two washes, cultures were suspended to 5 × 107 CFU/ml with physiological saline. 300 µl *S. suis* suspensions were mixed with 240 µl freshly undiluted and 1/500 diluted non-immune human serum and incubated at 37°C for 30 min. After two washes, *S. suis* was resuspended in 300 µl anti-C3b monoclonal antibody (20 µg/ml; Abcam, England) to detect C3 deposition and monoclonal anti-C5b-9 antibody (20 µg/ml; Abcam) to detect MAC formation. After incubation for 10 min at room temperature, FITC-conjugate goat-anti-mouse IgG (BD, USA) was used as the second antibody and incubated at room temperature for 10 min. Finally, bacteria were washed and resuspended in 800 µl physiological saline for flow cytometry analysis performed by FACSCalibur (BD). Bacteria were detected by log-forward and log-side scatter dot-plot at middle flow rate. A gating region was set to include the majority of bacteria except debris. 10,000 bacteria/events were acquired and analyzed for fluorescence using log-scale amplifications. C3 deposition and MAC formation capacities were measured by the geometric mean fluorescence intensity (GMFI). GMFI value was measured from three independent experiments performed in duplicate.

### SntA Immunodetection

Western-blot analysis was used to determine the location of SntA expressed in the SC-19, Δ*sntA*, and CΔ*sntA* strains. For cell lysate, cell cultures of *S. suis* strains were collected, resuspended with 1 ml bacterial lysis buffer (0.05 M Tris–HCl, 2.5 mM EDTA, 0.1 M NaCl, 0.25% Triton X-100, pH 8.5~9.0) and boiled 20 min to lysis, the resulting cell lysate were used to prepare SDS-PAGE samples. For cell wall proteins, samples were prepared as described previously (32). Briefly, bacterial cultures were collected; washed once in 50 mM Tris–HCl, pH 7.3; resuspended in 1 ml of osmoprotective buffer (50 mM Tris–HCl, pH 7.3, 20% sucrose, 2.5 µM PMSF) supplemented with 175 U/ml mutanolysin (Sigma-Aldrich, Shanghai, IL, USA); incubated at 37°C for 90 min under constant gentle agitation. After centrifugation at 12,000 *g*, 4°C for 15 min, supernatants containing the cell wall proteins were used to prepare SDS-PAGE samples. For secreted proteins, culture supernatants were collected, filtered (Millipore, 0.22 µm, Shanghai, China), and concentrated to 10 mg/ml, and then the proteins were sedimentated with pre-cooled 10% TCA-acetone solution at 4°C overnight. After centrifugation at 12,000 *g*, 4°C for 15 min, washed sediment with pre-cooled 90% acetone supplemented with 10 mM DTT for three times. The last sediment was resuspended with 8 M urea, 2% CHAPS, 10 mM DTT for next step. For all SDS-PAGE samples immunodetection, total proteins were quantified by BCA protein assay kit (Kangwei, Beijing, China), and then 48 µg cell lysate, secreted, and cell wall proteins were applied to 10% SDS-PAGE. Proteins transferred onto PVDF membrane (Millipore, 0.45 µm). Mouse SntA polyclonal antibody (24) was used for SntA immunodetection, and chemiluminescence detection (Bio-Rad, USA) with HRP-conjugate goat anti-mouse IgG (Antgene, Wuhan, China) by MF-Chemi BIS 3.2 (DNR, Israel).

### Preparation of Recombinant Proteins

Recombinant SntA protein (Protein ID: WP\_012027972.1) was expressed and purified as our previous report (24). To obtain the recombinant proteins of the complement C1q subunits, the cDNAs of C1qA, C1qB, and C1qC were amplified from the total cDNA of porcine lung tissue with primers C1qA\_F/R, C1qB\_F/R, C1qC\_F/R, respectively (**Table 1**). Total cDNA was prepared from total RNA of porcine lung tissue by using RT-PCR. Then the C1qA cDNA was cloned into pQE-32 plasmid (Qiagen, Shanghai, China), while the cDNAs of C1qB and C1qC were cloned into pET-28a-SUMO plasmid (26) to promote soluble expression. The resultant recombinant plasmids were transformed into *E. coli* BL21 (DE3), respectively. The expression of C1qA, C1qB, and C1qC proteins was induced by 1 mM isopropyl-β-d-thiogalactoside for 4 h at 37°C. The recombinant proteins were purified by using Ni-NTA agarose column (Bio-Rad) and quantified by BCA protein assay kit (Kangwei).

### Interaction Between C1q and SntA Proteins

ELISA was used to confirm the interaction between SntA and C1q proteins as described previously (33). For SntA bound assay, the proteins C1q (Quidel, USA), three C1q subunits C1qA, C1qB, C1qC, and BSA were coated to 96-well plates (BIOFIL, Guangzhou, China) with increasing concentrations of 0–7 µg/ml at 4°C for 16 h, respectively. After blocking with blocking buffer (PBS supplemented with 0.05% Tween20 and 1% BSA), 5 µg/ml SntA was poured to each well, and then incubated at 37°C for 1 h. Mouse SntA polyclonal antibody was applied at 37°C for 1 h to test the direct binding capacity. For C1q bound assay, SntA, BSA, and heme were coated to 96-well plates with increasing concentrations of 0–7 µg/ml at 4°C for 16 h, respectively, After blocking with blocking buffer, 5 µg/ml C1q was applied to each well at 37°C for 1 h. Rabbit C1qA polyclonal antibody (Abclonal, Wuhan, China) was applied at 37°C for 1 h to test the direct binding capacity. For inhibition assay, 5 µg/ml human aggregated IgG (Sigma-Aldrich) was coated to plates at 4°C overnight. Human aggregated IgG was prepared by incubating at 63°C for 20 min, immediately placing on ice for 1 h, centrifuging at 16,000 *g* for 5 min and quantification (34). C1q was pre-incubated with increasing concentrations of 0–25 µM SntA, heme, or BSA, and then applied to plates at 37°C for 1 h. Rabbit C1qA polyclonal antibody was applied to plates at 37°C for 1 h to test the indirect binding capacity. All the ELISA assays, the HRP-conjugate goat-anti-rabbit IgG (Antgene), or goat-anti-mouse IgG was used as the second antibody at 37°C for 1 h before chromogenic reaction.

Competitive binding assays were performed as described previously (35). For C1q-IgG binding assay, microtiter plates were pre-coated with 30 µg/ml human C1q overnight at 4°C. After blocking, plates were incubated with the mixtures of 50 µl 2.5 mg/ml human aggregated IgG and 50 µl increasing concentrations of 0–50 µM SntA or BSA in HB++ buffer (10 mM Hepes, pH 7.4, 100 mM NaCl, 5 mM CaCl2, 1 mM MgCl2). After incubation for 1 h at 37°C, plates were washed with washing buffer (HB++ buffer supplemented with 0.05% Tween20) and developed with HRP-conjugate goat anti-human IgG Fc (1:4,000; Sigma-Aldrich) at 37°C for 1 h before chromogenic reaction. For C1q–Ag–Ab binding assay, microtiter plates were coated with 2.5 µg/ml tetanus toxoid (Millipore) as an antigen for immune complex formation. After blocking, human tetanus toxin immunoglobulin (1:1,000; Hualan Bio, Xinxiang, China) was applied to the tetanus toxoid-coated plates to form immune complexes for 1 h at 37°C. 50 µl 2 µg/ml human C1q were mixed with 50 µl increasing concentrations of 0–50 µM SntA or BSA. The resulting mixtures were applied to each well at 37°C for 1 h. After washed, polyclonal goat antiserum to human C1q (1:5,000; Quidel, USA) was incubated to plates at 37°C for 1 h. HRP-conjugate rabbit anti-goat IgG (Antgene) was used as the second antibody at 37°C for 1 h before Chromogenic reaction. These data are presented as mean ± SD from three separate experiments.

### Binding of C1q to *S. suis*

Overnight cultures of the *S. suis* strains in TSB were diluted in 1:100 and grown to mid-log phase without agitation at 37°C. The cultures were washed and resuspended in DGHB++ buffer to 5 × 108 CFU/ml. *S. suis* suspensions were incubated with 0, 10, and 20 µg/ml C1q proteins at 37°C for 1 h and the total volume was 100 µl. After incubation and two washes, FITC-rabbit antihuman C1q antibody (Abcam) was applied for 30 min at 37°C. Finally, the bacteria was washed and resuspended in 500 µl PBS for flow cytometry analysis by FACSCalibur. Data from three independent experiments in duplicate were analyzed by Flow Jo 7.6.1 software (33). The assay was repeated three times.

### Hemolytic Assay

The hemolytic activity of SntA protein was measured as previous study (33). For the classical pathway, sheep red blood cells (SRBCs) (Baiji, Zhengzhou, China) were washed with pre-cooled DGHB++ buffer for three times and diluted to a concentration of 1 × 109 cells/ml. Then the SRBCs solution was mixed with equal volume of ambocepter (1:1,000; Baiji) and rotated at 37°C for 20 min. After two washes, 0.2% NHS was incubated with 0–100 µg/ml SntA at 37°C for 15 min, then the mixtures were incubated with 3 × 108 cells/ml SRBCs at 37°C for 1 h. BSA was used as a negative and the total volume is 150 µl. After centrifugation at 800 *g*, the hemolytic activity was calculated by spectrophotometric measurement of absorbance at 405 nm. For the alternative pathway, rabbit red blood cells (RRBCs) (Baiji) were washed with pre-cooled Mg2+-EGTA-DGHB buffer (4.2 mM Hepes, pH 7.4, 59 mM NaCl, 2.08% glucose, 0.08% gelatin, 7 mM MgCl2, 10 mM EGTA) for three times and diluted to a concentration of 1 × 109 cells/ml. 1.25% NHS was incubated with 0–100 µg/ml SntA at 37°C for 15 min, then the mixtures were incubated with 3 × 108 cells/ml RRBCs at 37°C for 1 h. The hemolytic activity of alternative pathway was determined as the classical pathway. These data are presented as mean ± SD from three separate experiments.

### Complement Activation Assay

To detect whether SntA could activate complement directly, complement activation ELISA were performed as described previously (33). 2 µg/ml SntA, 2 µg/ml BSA, 10 µg/ml IgM (Berseebio, Beijing, China) for the classical pathway, 100 µg/ml Mannan (Sigma) for the lection pathway, and 20 µg/ml Zymosan (Sigma) for the alternative pathway were coated to 96-well plates in PBS. The plates were washed with PBS supplemented with 0.05% Tween20 for each step. After blocking with the blocking buffer, 0–7% NHS in DGHB++ buffer was applied for classical pathway, 0–7% depleted of protein C1q serum (Quidel) in DGHB++ buffer was applied for lectin pathway, and 0–7% NHS in Mg2+-EGTA-DGHB buffer was applied for alternative pathway. After incubation for 1 h, the plates were washed and C3b rabbit polyclonal antibody (1:1,000; Proteintech, Wuhan, China) was poured to plates to detect C3 deposition at 37°C for 1 h. All the ELISA assays, the HRP-conjugate goat-anti-rabbit IgG was used as the second antibody at 37°C for 1 h before chromogenic reaction. These data are presented as mean ± SD from three separate experiments.

## RESULTS

### SntA Contributes to the Virulence of *S. suis*

Two mouse infection models were performed to investigate the contribution of *sntA* on virulence. The growth curves of *S. suis* presented by OD and CFU were measured before infection in mice. Results showed that no significant differences were observed in SC-19, Δ*sntA*, and CΔ*sntA* in both OD and CFU (Figure S2 in Supplementary Material). First, groups of eight mice were intraperitoneally infected with 2 × 109 CFU/mouse SC-19 and Δ*sntA*. Physiological saline was used as a negative control. Survival rate of infected mice was measured within 5 days post infection (dpi). We observed that four of mice infected with SC-19 died within 24 hpi, another four showed obvious limping, shivering, and central nervous system failure within 5 dpi. By contrast, only two mice infected with Δ*sntA* died within 24 hpi, one mouse died within 36 hpi, and another five mice were survived during the observation within 5 dpi (**Figure 1A**). This revealed that significant difference was observed in survival rate of SC-19 and Δ*sntA* infected mice. Second, groups of seven mice were intraperitoneally infected with the mixtures of SC-19 and Δ*sntA* at the ratio 1:1 (5 × 108 CFU/mouse). Blood and brain, lung colonization were recovered at 6, 12, 24, and 48 hpi, respectively. The efficiency of colonization of SC-19 was much higher at 24 and 48 hpi than that of Δ*sntA* in blood (**Figure 1B**) and lung (**Figure 1D**). In addition, significant differences were observed in brain from 6 to 48 hpi (**Figure 1C**). The results showed that SntA contributed to the survival in blood and the colonization in specific organs.

### SntA Possess Obvious Anti-Phagocytic Activity

We constructed a complementary strain of *sntA*, as confirmed by genome PCR (Figure S1A in Supplementary Material) and RT-PCR (Figure S1B in Supplementary Material). To investigate the anti-phagocytosis mediated by SntA, three bacteria killing assays and one phagocytosis assay were performed. In blood killing assay, the survival rate of SC-19 in human blood was 105%, but the absence of *sntA* decreased the survival rate to only 33% (**Figure 2A**). Similar results were observed in mouse blood killing assay (Figure S3A in Supplementary Material). In PMNs killing assay, PMNs supplemented with 20% active human serum were used to test the survival abilities of *S. suis* in human neutrophils. Results revealed that the survival rate of Δ*sntA* was significantly decreased compared to SC-19 and CΔ*sntA* (**Figure 2B**). In phagocytosis assay, RAW 264.7 cells were used to test anti-phagocytic activity of *S. suis*. Results demonstrated that the phagocytic numbers of Δ*sntA* were much higher comparing with SC-19 and CΔ*sntA* (**Figure 2C**). In the human serum killing assay, the active and inactivated human serum were used to investigate the role of complement on *S. suis* clearance. For the *sntA*-deleted mutant strain, only 48% viable Δ*sntA* bacteria survived in freshly active human serum. The presence of *sntA* significantly increased the survival rate of *S. suis* up to 250% (SC-19) and 185% (CΔ*sntA*), respectively. No obvious differences were observed in the survival rates of *S. suis* in inactivated serum (**Figure 2D**). Normal mouse serum was also used in this assay, and similar results were obtained (Figure S3B in Supplementary Material). No significant differences were observed between SC-19 and CΔ*sntA* in all tests. These data demonstrated that SntA was involved in resistance to phagocytosis and the bactericidal activity of blood, PMNs, and NHS dependent on complement. This reveled that SntA could obviously contribute to anti-phagocytosis and this may be involved in complement.

### SntA Inhibits the C3 Deposition and MAC Formation on *S. suis*

SntA possessed anti-phagocytosis, but the effect of SntA on complement activation on *S. suis* was still unclear. To investigate that, the C3 deposition and MAC formation assays were performed in both undiluted and 1:500 diluted human serum. Results showed that the viable bacteria number aggregated fluorescence of Δ*sntA* in C3 deposition was obviously increased compared to SC-19 and CΔ*sntA* in undiluted human serum (**Figures 3A,C**). The efficiency of MAC formation was significantly higher comparing with SC-19 and CΔ*sntA* (**Figures 3B,C**) as well. To assess the effect of SntA on complement activation through the classical pathway, 1/500 NHS was used. Results revealed that C3 deposition and MAC formation on surface of Δ*sntA* was significantly higher compared with SC-19 and CΔ*sntA* in 1/500 diluted

5 × 108 CFU/mouse). Results were shown as log10 of recovered bacteria counts after deletion of the highest and lowest value (CFU/ml in blood and CFU/g in organs). The statistical significance was shown by asterisks (unpaired *t* test; \*\*\**p* < 0.001; \*\**p* < 0.01; \**p* < 0.05; ns, *p* > 0.05).

human serum (**Figures 3D–F**). No significant differences were observed between SC-19 and CΔ*sntA* in all tests. These assays demonstrated that SntA could inhibit the complement pathway.

## SntA Is a Cell Wall Anchored Protein

The cell wall attached protein LPXTG motif region was found at C terminal of amino acid sequence of SntA (**Figure 4A**). To confirm this prediction, western-blot analysis was performed. Results showed that SntA expressed in cell lysate extracted from SC-19 and CΔ*sntA*, but not detected in Δ*sntA*. Cell wall proteins extracted by mutanolysin and secreted proteins sedimentated by TCA-acetone were used to further determine the location of SntA in *S. suis*. SntA was demonstrated to express in cell wall proteins, and not in secreted proteins (**Figure 4B**). These assays indicated that SntA existed in cell wall.

## SntA Interacts With Complement C1q

To assess the binding capacity between SntA and C1q, the direct and indirect bound assays were performed. In direct bound assay, C1q could significantly bind with SntA in a dose-dependent manner in both C1q-coated and SntA-coated plates (**Figures 5A,B**). The known C1q-binding molecule heme was used as a positive control and BSA as a negative control. In inhibition assay, both SntA and heme could significantly inhibit the binding capacity between plasma purified C1q and aggregated human IgG on IgGcoated plates, and the inhibitory effect of SntA was much stronger than the positive control heme (**Figure 5C**). Furthermore, three subunits of C1q including C1qA, C1qB, and C1qC could obviously interact with SntA in a dose-dependent manner as well (**Figure 5D**). These results indicated that recombinant SntA could bind to C1q. To investigate whether SntA bound with C1q on bacterial surface, flow cytometry was performed. A dosedependent increase of C1q-binding capacity was observed on *S. suis*. Compared with SC-19 and CΔ*sntA*, a significant reduction of C1q-binding capacity was observed in Δ*sntA* when 20 µg/ml C1q was added (**Figure 5E**). Taken together, SntA could interact with C1q and act as a C1q ligand on bacterial surface.

## SntA Competitively Bind With C1q

The classical pathway is triggered through the recognition of antigen-antibody complex on micro surface by recognition molecule C1q (4). To assess the effect of C1q-binding protein SntA on the recognition step of the classical complement pathway, competitive binding assays were performed. In C1q–IgG binding assay, result showed that SntA strongly decreased the C1q–IgG binding capacity, and 50 µM SntA inhibited the binding capacity down to 3.34% (**Figure 6A**). In C1q–Ag–Ab binding assay, the binding capacity was significantly decreased in a dose-dependent manner and 84.7%

\*\*\**p* < 0.001; \*\**p* < 0.01; \**p* < 0.05; ns, *p* > 0.05).

of it was remarkably inhibited with 50 µM SntA (**Figure 6B**). Results demonstrated that SntA could inhibit C1q–IgG binding and C1q– Ag–Ab binding by competitive interaction with C1q.

### SntA Inhibits Hemolytic Activity

SntA inhibited complement pathway, in which pathway SntA acted remain unclear. Hemolytic activity assay is often used to assess the activity of the classical pathway of complement activation with sheep erythrocytes (SRBCs) and of alternative pathway with rabbit erythrocytes (RRBCs) (33). So, to test in which pathway SntA inhibited complement, the hemolytic activity assays were performed. Results showed that SntA remarkably inhibited the hemolytic activity of SRBCs in a dose-dependent manner mediated by the classical pathway in the presence of NHS (**Figure 7A**). No inhibitory effect was observed in the negative control of BSA. However, SntA did not inhibit the hemolytic activity of RRBCs mediated by the alternative pathway (**Figure 7B**). These data revealed that SntA could inhibit the hemolytic activity mediated by the complement classical pathway.

### SntA Directly Activates Complement Pathways

To test whether SntA could activate complement directly, the complement activation assays were performed. In the classical pathway, the increasing concentrations of 0–7% NHS were applied to the SntA pre-coated plates to detect C3b deposition with specific C3b polyclonal antibody. IgM acted as a positive control and BSA as a negative control. Significant activation was observed on the SntA pre-coated plates and not on the BSA pre-coated plates (**Figure 8A**). This showed that SntA could activate complement through the classical pathway, although the activation level was lower than the positive control. In the lectin pathway, microtiter plates pre-coated with SntA and positive control Mannan exhibited strong complement activation mediated by the lectin pathway when human serum depleted of C1q was used (**Figure 8B**). Result demonstrated that SntA could activate the lectin pathway directly. In the alternative pathway, NHS in Mg2+-EGTA-DGHB buffer was used to block classical activation pathway and detect complement activation mediated by the alternative pathway. Result showed that no significant activation was observed in the plates pre-coated with SntA, while the positive control Zymosan obviously activated the alternative pathway (**Figure 8C**). Thus, SntA could directly activate the complement by the classical and lectin pathway, not the alternative pathway.

### DISCUSSION

Streptococcal toxic shock like syndrome, meningitis, and septicemia are the most important characteristics of the recently

by count of positive cell aggregated with fluorescence in the presence of complement (100 and 0.2% normal human serum). The representative histogram of flow cytometry from three independent experiments was exhibited (A,B,D,E). Geometric mean fluorescence intensity (GMFI) value was measured from three independent experiments performed in duplicate. The GMFI value of C3 deposition and MAC formation of SC-19 in all tests was set as 1.00, the corresponding Δ*sntA* and CΔ*sntA* values were proportional achieved. The resulting C3 deposition and MAC formation index were presented (C,F). The statistical significance was calculated by two-way ANOVA of Sidak's correct for multiple comparison and shown by asterisks (\*\**p* < 0.01; \**p* < 0.05; ns, *p* > 0.05).

Figure 4 | SntA was a conserved cell wall anchored protein. (A) Conserved domain of SntA was predicted in NCBI database (https://www.ncbi.nlm.nih.gov/cdd). SntA had two conserved domain: MPP\_CpdB\_N and 5′-nucleotid\_C, and a cell wall attached LPATG motif. (B) Western-blot was used to determine surface location of SntA. Cell lysate, cell wall proteins, and secreted proteins were ran 10% SDS-PAGE, transferred onto PVDF membrane and detected by mouse anti-SntA poly-antibody following by HRP-conjugate goat-anti-mouse IgG. WT, SC-19; MT, Δ*sntA*; CM, CΔ*sntA*.

emerging *S. suis* infection, especially in Southeast Asia. During infection, the pathogen must compete with the normal microflora, resist defense mechanisms of the local mucosal immunity, adhere and invade the mucosal epithelial cell barrier, subsequently reach and survive in the blood stream, and finally invade multiple organs including spleen, liver, kidney, and lung, even entry BBB (36). A plenty of factors have been demonstrated to be associated with these processes [reviewed in Ref. (37)]. However, how *S. suis* escape the immune surveillance to invade into blood and most organs is largely unknown. In this study, we found that the surface heme-binding protein SntA of *S. suis* (24) contributes to the resistance of C3 deposition and MAC formation on bacterial surface, the abilities of anti-phagocytosis, survival in blood, and *in vivo* colonization. SntA can interact with C1q, and inhibit the hemolytic activity *via* the classical pathway. SntA could also directly activate classical and lectin pathways, resulting in complement consumption. These two complement evasion strategies may be crucial for the pathogenesis of the zoonotic pathogen *S. suis*.

In our previous study, SntA is identified as a heme-binding protein which can interact with the host antioxidant protein AOP2 and inhibit its antioxidant activity, and thus contribute to the survival and pathogenesis of *S. suis* in pigs (24). This protein is a bifunctional 2′,3′-cyclic nucleotide 2′-phosphodiesterase/ 3′-nucleotidase, which is distributed in many species of Grampositive bacteria (24). In the present study, mouse infection model is used to further study the function of SntA involving

plates to test the SntA bound capacity. (E) Increasing concentrations of C1q were incubated with *Streptococcus suis* to detect the binding capacity by FITCanti-human C1q antibody. Results were expressed from three independent experiments performed in triplicate. The statistical significance was calculated by two-way ANOVA of Sidak's correct for multiple comparison and shown by asterisks (\*\*\*\**p* < 0.0001; \*\*\**p* < 0.001; \*\**p* < 0.01; \**p* < 0.05; ns, *p* > 0.05).

Figure 6 | SntA competitively binds with C1q. (A) Inhibition of IgG–C1q binding. Increasing concentrations of SntA were premixed with C1q and applied to IgG-coated plates to test the inhibitory capacity of SntA to C1q–IgG binding. (B) Inhibition of C1q–Ag–Ab binding. IgG was applied to tetanus toxoid-coated plates for IgG-containing immune complex formation. Increasing concentrations of SntA were premixed with C1q and added to plates to test the inhibitory capacity of SntA to C1q–Ag–Ab binding. BSA was served as a negative control. Results were expressed from three independent experiments performed in triplicate. The statistical significance was calculated by two-way ANOVA of Sidak's correct for multiple comparison and shown by asterisks (\*\*\*\**p* < 0.0001; ns, *p* > 0.05).

in the pathogenesis. Results show that SntA facilitates in *S. suis* survival in blood (**Figure 1B**) and subsequent colonization in some specific organs (**Figures 1C,D**). To entry the bloodstream and spread to distant organs, the Gram-positive pathogens usually utilize opsonophagocytic clearance function of neutrophils and macrophages (38). SntA is demonstrated to contribute to the anti-phagocytosis of *S. suis* mediated by neutrophils and macrophages and the survival in whole blood and serum *in vitro*

Figure 7 | SntA inhibits hemolytic activity. (A) To measure the inhibitory effect of SntA on the classical pathway, antibody-coated sheep erythrocytes were subjected to complement attack from normal human serum (NHS) in the presence of the increasing concentrations of SntA. BSA was used as a negative control. The degree of lysis was estimated by measurement of the release of hemoglobin. (B) To measure the inhibitory effect of SntA on the alternative pathway, rabbit erythrocytes were subjected to complement attack from NHS in the presence of the increasing concentrations of SntA. BSA was used as a negative control. Cell lysis was measured like (A). The absorbance without inhibitor was set to 100%. Results were expressed from three independent experiments performed in triplicate. The statistical significance was calculated by two-way ANOVA of Sidak's correct for multiple comparison and shown by asterisks (\*\*\*\**p* < 0.0001; \*\**p* < 0.01; ns, *p* > 0.05).

the plates pre-coated with SntA, Zymosan as a positive control to detect C3b deposition. Results were expressed from three independent experiments performed in triplicate. The statistical significance was calculated by two-way ANOVA of Sidak's correct for multiple comparison and shown by asterisks (\*\*\*\**p* < 0.0001; \*\*\**p* < 0.001; \*\**p* < 0.01; \**p* < 0.05; ns, *p* > 0.05).

(**Figure 2**). The efficient phagocytosis is an important innate immune mechanism for controlling infection of extracellular bacteria such as *Streptococci*, and this process requires serum with intact complement activity (39). Recently, many complement inhibitors target at complements such as FH (29), C1q (23, 40), C3 (41), C4BP (42), mediate complement evasion, and subsequently contribute to pathogenesis in Gram-positive bacterium. In order to investigate whether SntA involves in the complement evasion of *S. suis*, a series of experimentations are carried out to this end.

First, the complement deposition assays are performed. The C3 deposition and MAC formation on the surface of the mutant strain Δ*sntA* are significantly higher than those on the parental strain SC-19 and complementary strain CΔ*sntA* (**Figure 3**). This indicates that SntA involved in complement activation by *S. suis*. The undiluted and 1:500 diluted human serum (NHS) are used in the complement deposition assays, respectively, C3 deposition and MAC formation on the surface of Δ*sntA* are always significantly higher than those on SC-19 and CΔ*sntA* (**Figure 3**). This suggests that SntA could inhibit both classical pathway and alternative pathway because it has been documented that the classical pathway is activated by using diluted NHS (35), whereas alternative pathway is probably activated by using undiluted NHS in the complement activation assays (30).

Hemolytic assay is often used to assess the activation of the classical pathway by using sheep erythrocytes and the activation of alternative pathway by using rabbit erythrocytes (33). To confirm which complement pathway is inhibited by SntA, hemolytic assays have been performed using recombinant SntA protein. The results show that SntA could inhibit the hemolytic activity of SRBCs, but not of rabbit erythrocytes (**Figure 7**). This indicates that SntA could only inhibit the classical pathway. Furthermore, SntA is confirmed as a cell wall anchored protein that could interact with C1q in a dose-dependent manner (**Figures 4** and **5**). ELISA experiments also suggest that SntA could interact with the three C1q subunits C1qA, C1qB, and C1qC, respectively (**Figure 5**). It is very interesting whether this interaction results in the complement evasion. To this end, the competitive binding assays have been done by ELISAs reported previously (35). The results show that SntA could competitively bind C1q with IgG and Ag–Ab complex (**Figure 6**). Thus, SntA can interact with C1q and inhibit the hemolytic activity mediated by the classical pathway, consequently contribute to complement evasion. Similar inhibitory mechanism has been identified in other Gram-positive strain. *Streptococcus pneumococcal* (33) and *S. aureus* (23) infection. These bacterial proteins interact with C1q and specially inhibit the classical complement pathway, consequently facilitate *pneumococcal* complement escape (23, 33).

Concerning that C1q is a complex of 18 polypeptide chains consisting of six C1qA, C1qB, and C1qC subunits, the binding assays using each subunit could not properly reveal the real interactions between SntA and C1q although it has been reported that the globular heads of the recombinant subunits can assemble to form the high molecular weight oligomers (43). Interestingly, heme has also been demonstrated to inhibit the classical pathway through binding with C1q (35), and SntA is a heme-containing protein (24), it warrants further investigation whether the heme in SntA or SntA as a hemeprotein involves in the complement evasion. To this end, we are trying to obtain the recombinant SntA without heme and holo-form SntA although this is not an easy thing.

To look at whether SntA can directly activate complement pathways, the recombinant SntA protein is subjected to activate the complement C3. The results show that SntA could directly activate both classical and lectin pathways but not alternative pathway (**Figure 8**). This indicates that SntA could mediate complement evasion through complement consumption as well (33). In *S. pneumococcal*, PepO can activate complement through the classical and alternative pathway (33), while anther complement inhibitor phosphoglycerate kinase cannot activate complement (44). Research about complement evasion mediated by complement consumption is not much. The mechanism of this process warrants further study.

In conclusion, *S. suis* cell wall anchored heme-binding protein SntA mediates complement evasion by inhibition of complement activation and complement consumption. The inhibition of complement activation may be *via* the SntA–C1q interaction. These two complement evasion strategies may be crucial for the pathogenesis of *S. suis*.

### ETHICS STATEMENT

Animal experiments were approved by the Laboratory Animal Monitoring Committee of Huazhong Agricultural University and performed strictly according to the recommendations in the Guide for the Care and Use of Laboratory Animals of Hubei Province, China. Venous blood samples were provided by healthy donors and collected in accordance with the approved guidelines. Approval was obtained from the Institutional Medical Ethics Committee of Huazhong Agricultural University and the healthy donors provided written informed consent in accordance with the Declaration of Helsinki.

### AUTHOR CONTRIBUTIONS

The experiments were performed mainly by SD, some experiment material and data were provided by TX and LY, and some experiments were performed with the assistance of TX, QF, JZ, LC, and JL. SD analyzed the data. The study was conceived and designed by SD and RZ. SD and RZ wrote the manuscript.

### ACKNOWLEDGMENTS

The authors thank Dr. Qi Huang from University of Dundee in United Kingdom for revising the grammar in this manuscript and Dr. Takamatsu for providing the *S. suis*- *E. coli* shuttle cloning vector pSET2.

### FUNDING

This work was supported by the Natural Science Foundation of China (NSFC; 31472202), the National Key R&D Program of China (2017YFD0500201), and Hubei Province Natural Science Foundation for Innovative Research Groups (2016CFA015).

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at https://www.frontiersin.org/articles/10.3389/fimmu.2018.01063/ full#supplementary-material.

Figure S1 | Identification of *Streptococcus suis* strains SC-19, Δ*sntA*, and CΔ*sntA*. The *sntA* gene and its flanked genes (B9H01\_10065 and B9H01\_10075) were amplified by genome PCR (A) and reverse transcription-PCR (B) using primers SntA\_F/R, 10065\_F/R, and 10075\_F/R, respectively. WT, wild-type strain SC-19; MT, *sntA* gene mutant strain Δ*sntA*; CM, complementary strain CΔ*sntA*.

Figure S2 | The growth abilities of *Streptococcus suis* strains SC-19, Δ*sntA*, and CΔ*sntA*. (A) Bacterial cell density was measured by spectrophotometer at

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Figure S3 | SntA possess anti-phagocytic activity. (A) Survival rate of *Streptococcus suis* in mouse blood. (B) Survival rate of *S. suis* in mouse active and inactivated serum. Results were expressed from three independent experiments performed in triplicate. The statistical significance was showed by asterisks (unpaired *t* test; \*\*\**p* < 0.001; \*\*p < 0.01; \*p < 0.05; ns, p > 0.05).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Deng, Xu, Fang, Yu, Zhu, Chen, Liu and Zhou. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Plasminogen activator inhibitor 1 for Predicting Sepsis Severity and mortality Outcomes: a Systematic Review and meta-analysis

*Timothy L. Tipoe1,2, William K. K. Wu2,3\*, Lilianna Chung 4 , Mengqi Gong 5 , Mei Dong 6 , Tong Liu 5 , Leonardo Roever 6 , Jeffery Ho 2,3, Martin C. S. Wong7 , Matthew T. V. Chan 3 , Gary Tse1,2\*, Justin C. Y. Wu1 \* and Sunny H. Wong1,2\**

#### *Edited by:*

*Thierry Roger, Center Hospitalier Universitaire Vaudois (CHUV), Switzerland*

#### *Reviewed by:*

*Andreja Sinkovicˇ , University Clinical Center Maribor, Slovenia Per Morten Sandset, University of Oslo, Norway*

#### *\*Correspondence:*

*William K. K. Wu<EMAIL_ADDRESS>Gary Tse<EMAIL_ADDRESS>Justin C. Y. Wu<EMAIL_ADDRESS>Sunny H. Wong<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 02 March 2018 Accepted: 15 May 2018 Published: 18 June 2018*

#### *Citation:*

*Tipoe TL, Wu WKK, Chung L, Gong M, Dong M, Liu T, Roever L, Ho J, Wong MCS, Chan MTV, Tse G, Wu JCY and Wong SH (2018) Plasminogen Activator Inhibitor 1 for Predicting Sepsis Severity and Mortality Outcomes: A Systematic Review and Meta-Analysis. Front. Immunol. 9:1218. doi: 10.3389/fimmu.2018.01218*