Preserving vascular integrity protects mice against multidrug- resistant gram-negative bacterial infection

Article abstract of DOI:10.1128/AAC.00303-20

The rise in multidrug-resistant (MDR) organisms portends a serious global threat to the health care system with nearly untreatable infectious diseases, including pneumonia and its often fatal sequelae, acute respiratory distress syndrome (ARDS) and sepsis

Full text of DOI:10.1128/AAC.00303-20

AAC Accepted Manuscript Posted Online 11 May 2020
Antimicrob. Agents Chemother. doi:10.1128/AAC.00303-20
Copyright © 2020 American Society for Microbiology. All Rights Reserved.
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Preserving Vascular Integrity Protects Mice Against Multidrug-Resistant
Gram-Negative Bacterial Infection
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Teclegiorgis Gebremariam, Lina Zhang, Sondus Alkhazraji, Yiyou Gu, Eman G. Youssef,
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Zongzhong Tong, Erik Kish-Trier, Ashok Bajji, Claudia V. de Araujo, Bianca Rich, Samuel
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W. French, Dean Y. Li,
Alan L. Mueller, Shannon J. Odelberg, Weiquan Zhu, and Ashraf
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S. Ibrahim,
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The Lundquist Institute for Biomedical Innovations at Harbor-University of California Los
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Angeles (UCLA) Medical Center, Torrance, California, USA; College of Wildlife Resources,
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Northeast Forestry University, Harbin, China; Faculty of Postgraduate Studies for Advanced
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Sciences, Beni-Suef University, Beni-Suef, Egypt; A6 Pharmaceuticals, Salt Lake City, Utah,
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USA; Department of Internal Medicine, University of Utah, Salt Lake City, Utah, USA; David
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Geffen School of Medicine at UCLA, Los Angeles, California, USA; Departments of Human
Genetics and Oncological Sciences, University of Utah, Salt Lake City, Utah, USA.
Running title: ARF6 inhibition and Gram negative bacterial infection
Word count: 3,803
‡
Address Correspondence: Prof. Ashraf S. Ibrahim. David Geffen School of Medicine.
Division of Infectious Diseases, Horbor-UCLA Medical Center. E-mail:
ibrahim@lundquist.org. Tel: 310-222-6424. Fax: 310-803-5620.
^Currently employed by Merck
Key words: Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumonia, ARF6,
LPS, drug resistance, sepsis, Gram-negative bacteria
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ABSTRACT
The rise in multidrug resistant (MDR) organisms portends a serious global threat to the
healthcare system with nearly untreatable infectious diseases, including pneumonia and its often
fatal sequelae, acute respiratory distress syndrome (ARDS) and sepsis. Gram-negative bacteria
(GNB) including Acinetobacter baumannii, Pseudomonas aeruginosa, and carbapenemase-
producing Klebsiella pneumoniae (CPKP), are among the World Health Organization and National
Institutes of Health’s high priority MDR pathogens for targeted development of new therapies. Here
we show that stabilizing the host’s vasculature by genetic deletion or pharmacological inhibition
of the small GTPase ADP-ribosylation factor 6 (ARF6) increases survival rates of mice infected
with A. baumannii, P. aeruginosa, CPKP pneumonia. We show that pharmacological inhibition
of ARF6-GTP phenocopies endothelial-specific Arf6 disruption in enhancing survival of mice
with A. baumannii pneumonia, suggesting that inhibition is on target. Finally, we show that the
mechanism of protection elicited by these small molecule inhibitors is by restoration of vascular
integrity disrupted by GNB lipopolysaccharide (LPS) activation of TLR4/MyD88/ARNO/ARF6
pathway. By targeting the host’s vasculature with small molecule inhibitors of ARF6 activation,
we circumvent microbial drug resistance and provide a potential alternative/adjunctive treatment
for emerging and re-emerging pathogens.
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INTRODUCTION
Current global trends in healthcare-associated infections (HAIs) are characterized by the
emergence and re-emergence of MDR pathogens (1). There are ~1.0 million HAIs in the
US/year, which cause 70,000-100,000 deaths and $30B in costs (2). GNB are responsible for half
of all HAIs. In particular, Acinetobacter baumannii, Pseudomonas aeruginosa, and
carbapenemase-producing Klebsiella pneumoniae (CPKP) have emerged as predominant causes of
MDR HAIs (3, 4). Some strains of these pathogens are resistant to all FDA-approved antibiotics
and in many cases lead to serious complications (e.g., sepsis (5)), which are associated with high
mortality and morbidity rates (6-8). Sepsis results from a dysregulated host immune response
associated with production of a cytokine storm, which contributes to increased vascular
permeability, tissue edema, organ failure, and death (9-12).
The past few decades provide evidence that development of resistance to antibiotics is
inevitable. Thus, it is essential to develop novel treatments that do not rely solely on antibacterial
action. Altering the patient’s own host response is one such alternative strategy for combating
infectious diseases. Stabilizing the vasculature to prevent fluid leak, without compromising the
beneficial innate immune system response, may allow the host ample time to clear the infection
without causing organ failure. The activation state of ARF6, which is a convergence point in the
signaling pathways of several inflammatory mediators and cytokines, plays a major role in
controlling vascular permeability (13-16). When ARF6 is in its activated GTP-bound state,
permeability is increased, and when it is in its inactive GDP-bound state, the vasculature is
stabilized leading to decreased permeability. Proinflammatory agonists such as IL-1β, GNB
lipopolysaccharides (LPS), and vascular endothelial growth factor (VEGF) activate ARF6, which
in turn promotes the internalization of the adherens junction protein VE-cadherin and thereby
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increases endothelial permeability (13, 15, 16). We have shown that stabilizing the vasculature
by reducing ARF6 activity is an effective strategy for reversing pathology and increasing
survival rates in several preclinical models of inflammatory and vascular disease, including
arthritis (15), LPS-induced endotoxemia (13), and diabetic retinopathy (16). Here, we show that
stabilizing the vasculature through pharmacological inhibition of ARF6 activation by small
molecules can increase survival rates and reduce pathology in preclinical models of MDR GNB
infections.
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RESULTS AND DISCUSSION
Arf6 conditional knockout mice are resistant to A. baumannii pneumonia
We previously showed that mice infected with MDR A. baumannii die from endotoxemia
caused by LPS activation of Toll Like Receptor-4 (TLR4) since TLR4-deficient mice are
completely protected from A. baumannii bacteremia (17). We hypothesized that triggering of
TLR4 by LPS during A. baumannii infection leads to activation of ARF6, resulting in increased
virulence by compromising vascular integrity. To test this hypothesis, we compared the virulence
of A. baumannii HUMC1 (a strain resistant to all antibiotics except colistin) (18, 19) in wild-type
versus Arf6 conditional knockout mice using pulmonary infection. Arf6 conditional knockout
mice were significantly more resistant to A. baumannii pneumonia with 40% overall survival
f/+
after 21 days post infection versus 0% survival for mice carrying two active Arf6 alleles (Arf6 )
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f/-
(Fig. 1A). In another study, Arf6 or Arf6 conditional knockout mice (Arf6 ; +/Tie2-cre) were
infected with A. baumannii via inhalation and sacrificed for lung bacterial burden 4 days post
infection. Arf6 knockout mice had ~1.5 log10 lower A. baumannii burden than wild-type mice
(Fig. 1B). Collectively, these studies suggest that disruption of endothelial cell ARF6 allows the
mouse immune system the opportunity to clear A. baumannii infection.
To test whether enhanced resistance of Arf6 knockout mice to A. baumannii pneumonia is
due to enhanced vascular integrity of the lungs, we infected mice with A. baumannii as above
and 3 days later intravenously injected them with Evans blue dye to assess lung permeability. As
f/+
expected, Arf6 mice showed increased vascular permeability by ~25% when compared to Arf6
conditional knockout mice (Fig. 1C). Histopathological examination of the lungs corroborated
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the permeability results, with Arf6 mouse lungs demonstrating enhanced tissue edema with
100 extensive hemorrhage compared to Arf6 conditional knockout mice (Fig. 1D).
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02 ARF6 inhibitors are protective against murine bacterial pneumonia
03 Because ARF6 activation promotes virulence of A. baumannii, we hypothesized that
04 pharmacologic ARF6 inhibition could be used to target GNB infections by stabilizing the
05 vasculature. Thus, we tested whether pharmacologic inhibition of ARF6 could increase survival
06 of mice with MDR GNB pneumonia. Small molecule ARF6 inhibitors, NAV-2729 (structure and
07 method of synthesis were previously described in Yoo et al. (20)), A6-4424, and its prodrug A6-
08 5093 (Fig S1A) were evaluated for efficacy in treating murine pneumonia due to GNB. NAV-
09 2729 (IC50 = 1.5 M) has been shown to have therapeutic benefit in alleviating animal models
10 of diabetic retinopathy (16). A6-5093 is a new highly soluble prodrug of A6-4424 designed to
11 enhance in vivo administration (see Supplemental Methods for synthesis of A6-4424 and A6-
12 5093). When A6-5093 is administered in vivo, its lysyl group is rapidly cleaved to produce A6-
13 4424 (Fig. S1A), an active ARF6 inhibitor with IC50 = 1.9 M (Fig. S1B-C).
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Immunosuppressed mice were infected with aerosolized A. baumannii HUMC1 (a
15 virulent MDR strain of A. baumannii to which immunocompetent mice are resistant) (18, 19).
16 Mice were treated daily for 7 days with: 1) 30 mg/kg of NAV-2729 or A6-4424 (both dissolved
17 in DMA/PEG and the dose chosen based on pharmacokinetic (PK) data shown in Fig. S1D-G);
18 2) 43 mg/kg of A6-5093 (dissolved in saline and equivalent to 30 mg/kg of A6-4424); 3)
19 DMA/PEG (vehicle); or 4) 2.5 mg/kg bid of colistin. Vehicle-treated mice had a median survival
20 time of 8 days and almost complete mortality by Day 15 post infection. In contrast, NAV-2729
21 or A6-5093 enhanced median survival time to >21 days with an overall survival of >80% by Day
22 21 post infection (Fig. 2A). Although mice treated with A6-4424 had a median survival time
23 similar to vehicle-treated mice, 21-day survival was 50% versus 5% in vehicle-treated mice.
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24 Interestingly, the enhanced survival of mice seen with the ARF6 inhibitors far exceeded survival
25 seen with the current first-line therapy of colistin (Fig. 2A).
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We investigated if the survival benefit correlated with reduction of bacterial burden in the
27 lungs. Mice were infected and treated as above and sacrificed on Day 4 post infection. Mice
28 treated with NAV-2729 or A6-5093 had at least 1-log10 reduction in lung bacterial burden
29 compared to vehicle-treated mice (Fig. 2B). Since A. baumannii HUMC1 is colistin sensitive
30 (19), mice treated with colistin had a 2-log10 reduction of lungs bacterial burden versus vehicle-
31 treated mice and 1-log10 reduction versus ARF6 inhibitors (Fig. 2B). Thus, the decreased
32 efficacy of colistin in survival studies is likely attributable to its toxicity (21).
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We also evaluated the activity of A6-5093 against murine pneumonia caused by MDR P.
34 aeruginosa or CPKP. Mice infected with CPKP or P. aeruginosa and treated with A6-5093 had
35 70% and 45% 21-day survival rates versus 10% and 0% survival rates for vehicle-treated mice,
36 respectively (Fig. 2C and 2D). Collectively, these results confirm the protective activity of ARF6
37 inhibitors against at least three MDR GNB, which are noted for causing HAI (22).
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139 ARF6 inhibitors enhance vascular integrity without affecting inflammation
140 We investigated the mechanism by which ARF6 inhibitors protect from GNB infection. First,
141 we determined if any of our ARF6 inhibitors has a direct inhibitory or microbicidal effect on the
142 three MDR bacteria under study. We performed minimal inhibitory concentration testing of the
143 pathogen using NAV-2729, A6-4424, or vehicle control before and after using the bacteria in
144 infection models. We observed no difference in A. baumannii, CPKP, or P. aeruginosa growth
145 even at concentrations of 156 µM (data not shown), thereby indicating that the in vivo protection
146 is likely due to an effect of the inhibitors on the host.
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ARF6 activation leads to increased vascular permeability via internalization of the
48 adherens junction protein VE-cadherin (13, 15, 16). Thus, we hypothesized that ARF6 inhibitors
49 protect mice from GNB infection by maintaining vascular integrity. To investigate this
50 possibility, we determined the effect of ARF6 inhibitors on in vivo organ permeability and the
51 inflammatory response to A. baumannii pneumonia. Mice with A. baumannii pneumonia had an
52 ~50% and 30% increase in lung and kidney permeability, respectively, when compared to
53 uninfected mice. Treatment with either NAV-2729 or A6-5093 reduced the A. baumannii-
54 mediated vascular permeability of lungs and kidneys to levels seen with uninfected mice (Fig.
55 3A). These results were corroborated by histological examination of lungs harvested from mice
56 infected with A. baumannii, which showed signs of pneumonia and tissue edema consistent with
57 increased vascular permeability. Lungs harvested from mice infected and treated with either
58 NAV-2729 or colistin showed more normal architecture with minimal to no signs of tissue
59 edema (Fig. 3B). Necrobiosis was evident in spleens taken from A. baumannii-infected mice
60 without treatment or with those treated with colistin but not in spleens harvested from infected
61 mice and treated with NAV-2729 (Fig. 3B). The observed necrosis in spleens from colistin-
62 treated mice is likely attributable to toxicity of colistin (21).
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LPS-induced endotoxemia results in a robust immune response via TLR4-mediated NF-
64 κB activation (17). To determine the effect of ARF6 inhibitors on the inflammatory immune
65 response to A. baumannii pneumonia, we infected immunosuppressed mice and treated them as
66 described above for 4 days. Mice were sacrificed and plasma and lungs were collected for
67 determination of inflammatory cytokines. Due to the immunosuppression, which results in ~9
68 days of leukopenia (23), only IL-6 was detectable. No differences in IL-6 levels were observed
69 in the lungs or plasma collected from vehicle-, NAV-2729-, or colistin-treated mice (Fig. 3C).
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70 Collectively, these results suggest that ARF6 inhibitors likely protect mice from A. baumannii
71 pneumonia by preserving vascular integrity without affecting the inflammatory immune
72 response.
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174 Bacterial LPS induces vascular permeability via the MyD88/ARNO/ARF6-GTP pathway
175 Previously we showed that that TLR4 activation by LPS compromises vascular integrity via
176 activation of the MyD88/ARNO/ARF6 pathway (13). It was also shown that lethality in mice
177 infected with A. baumannii is mainly determined by the ability of the bacteria to activate TLR-4
178 through shed LPS. We hypothesized that GNB induces vascular permeability by activation of
179 the MyD88/ARNO/ARF6 pathway through their LPS. To investigate this hypothesis, we studied
180 if A. baumanaii and its LPS induce HUVEC vascular permeability in vitro. The virulent A.
181 buamanii MDR HUMC1 and its cell-free supernatant (this strain sheds LPS (17)) induced
182 HUVEC permeability equivalent to permeability seen with commercially available E. coli LPS,
183 while the avirulent drug-sensitive strain ATCC 17978 cells and supernatant did not (this strain
184 does not shed LPS (17)) (Fig. 4A). To confirm that the enhanced HUVEC permeability
185 observed following treatment with HUMC1 supernatant was due to LPS shedding, we removed
186 LPS using polymyxin B agarose prior to incubating HUVECs with the supernatant (Fig. S2).
187 Removal of LPS from the HUMC1 supernatant reduced permeability to uninfected HUVECs
188 levels (Fig. 4A), thus confirming the role of bacterial LPS in compromising vascular integrity.
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Mice with a TLR4 mutation or TLR4 knockout mice are resistant to A. baumannii
90 infection (17). Thus, we hypothesized that blocking TLR4 would abrogate the ability of A.
91 baumannii to induce HUVEC permeability. Indeed, anti-TLR4 antibodies completely blocked
92 the ability of A. baumannii HUMC1 or its cell-free culture supernatant to induce HUVEC
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193 permeability (Fig. 4B). Finally, by using ARF6-GTP pulldown assay (Fig. 4C), we show that A.
194 baumannii activated HUVEC ARF6 and NAV-2729 or A6-4424 reversed this activation and its
195 sequelae of endothelial permeability (Fig. 4D). Similarly, A6-5093 reduced CPKP- or P.
196 aeruginosa-induced HUVEC permeability (Fig. S3).
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ARF6 activation can be induced by multiple mechanisms including MyD88/ARNO (via
98 TLR4 or the IL-1 receptor), the TNF receptor, or the VEGF receptor (12-15). Since LPS-TLR4
99 interactions are critical for A. baumannii virulence, we hypothesized that activation of ARF6
00 occurs through stimulation of the MyD88/ARNO pathway. Consequently, downregulation of the
01 expression of MyD88, ARNO, or ARF6 should protect HUVECs from A. baumannii-induced
02 permeability. We used siRNA targeting MyD88, ARNO, or ARF6 to detect the contribution of
03 these proteins to A. baumannii-induced vascular permeability. Downregulating the expression of
04 MyD88 or ARNO by 70-85% (Fig. 5A) resulted in ~50% reduction in HUVEC ARF6-GTP
05 levels in HUVECs infected with A. baumannii (Fig. 5B). As expected, transfecting HUVECs
06 with siRNA targeting MyD88, ARNO, or ARF6 almost completely protected them from A.
07 baumannii-induced permeability (Fig. 5C).
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Collectively, these data show that vascular permeability is induced by GNB LPS via
09 TLR4 activation of the MyD88/ARNO/ARF6 pathway and that ARF6 inhibitors minimize GNB-
10 induced vascular leak in vitro.
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12 A. baumannii enhances HUVEC permeability by promoting internalization of VE-cadherin.
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13 We previously showed that LPS compromises vascular integrity by disruption of adherens
14 junctions via internalization of VE-cadherin (13). To investigate whether a similar mechanism
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215 might be driving vascular permeability during A. baumannii infection, A. baumannii HUMC1-
216 infected HUVECs were incubated with or without NAV-2729 and then immunostained with VE-
217 cadherin antibody without permeabilization. A. baumannii reduced VE-cadherin surface
218 expression at the cell-cell junctions (Fig. 6A) and resulted in disrupting the monolayer by 25%.
219 In contrast, addition of NAV-2729 to infected HUVECs preserved the surface expression of VE-
220 cadherin and maintained the integrity of the monolayer to levels similar to those seen with
221 uninfected HUVECs (Fig. 6A-C). Thus, A. baumannii compromises vascular integrity by
222 causing VE-cadherin internalization subsequent to ARF6 activation.
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Based on these data and previously published results (13-16), we propose a model of
24 MDR GNB virulence that relies on LPS binding to TLR4, which activates two independent
25 pathways that diverge at MyD88: 1) a pathway that activates a robust immune response via NF-
26 κB, which is characterized by an increase in inflammatory cytokines; and 2) a pathway that
27 compromises vascular integrity via activation of ARF6 and internalization of VE-cadherin,
28 resulting in excessive vascular leak, tissue edema, organ failure, and death. ARF6 inhibitors
29 block vascular leak without affecting the LPS-induced inflammatory immune response, thereby
30 allowing the immune system to fight the infection (Fig. 7).
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31
Population-based studies in high income countries predicated a global burden of sepsis of
32 ~31.5 million, of which 19.4 million are classified as severe sepsis cases resulting in an
33 estimated 5.3 million deaths annually (24). There have been >100 clinical trials to evaluate
34 potential therapies for sepsis in the last few decades. Strategies in these trials included the use of
35 non-selective inhibitors of inflammation (e.g. corticosteroids and non-steroidal anti-
36 inflammatories), neutralization of microbial mediators of sepsis such as endotoxin, and specific
37 inhibitors of host inflammatory mediators such as TNF-α, IL-1, and nitric oxide. Other strategies
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238 included the use of activators of immune function (e.g. G-CSF and interferon-Ɣ) and the
239 administration of anticoagulant molecules such as activated protein C (APC), tissue factor
240 pathway inhibitor (TFPI), anti-tissue factor antibody, anti-thrombin, thrombomodulin, or heparin
241 (Reviewed in (25)). Despite the promising preclinical data on these targets, none of the
242 conducted clinical trials identified a definitive treatment for sepsis. Importantly and despite the
243 lack of improvement in survival of sepsis patients, in aggregate these studies showed small but
244 consistent signal in clinical benefit (25). The failure of these clinical trials could be attributed to
245 the complexity of sepsis because it is a dysregulated host immune response modulated by
246 multiple factors and targeting one molecule is unlikely to show significant benefit unless the
247 target is known to control multiple processes that are activated during sepsis (e.g. targeting a
248 transcription factor that controls several of the inflammatory mediators) (25, 26). Alternatively, a
249 desirable target could be a convergent downstream effector that controls a process that is critical
250 in worsening the outcome of sepsis such as compromising vascular integrity (26). In this respect,
251 inhibition of the ARF6 molecule, which is a common convergence point in the signaling
252 pathways of several inflammatory mediators and cytokines (e.g. LPS, TNF-α, IL-1β, and VEGF)
253 (12, 13, 15, 16), is an attractive target in treating sepsis via maintaining vascular integrity.
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When designing clinical trials to evaluate potential treatments for sepsis, it is important to
55 endeavor to recruit a homogenous cohort of patients. This process should be guided by the
56 success and failure in preclinical models of infection. For example, in studying TNF-α as a
57 target, preclinical models suggest that TNF neutralization in experimental sepsis is most
58 efficacious in models of systemic endotoxemia or GNB infections, ineffective in the cecal
59 ligation and puncture model of polymicrobial sepsis, and even harmful in experimental models
60 of S. pneumoniae, Candida, Listeria, or M. tuberculosis infections (25). Our results indicate that
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261 ARF6 inhibition is more likely to be effective in patients infected with GNB sepsis rather than
262 those infected with polymicrobial organisms since A6-5093 was marginally effective in the cecal
263 ligation and puncture model (data not shown).
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In summary, by using ARF6 inhibitors to target the vascular leak that often accompanies
65 severe infections, we have markedly increased survival rates in preclinical models of life-
66 threatening infections of GNB. A primary advantage of this approach is that vascular leak is a
67 host response to infection. Therefore, targeting it should not lead to antimicrobial resistance. This
68 approach may prove to be applicable to any infection that activates MyD88/ARF6 (e.g.,
69 Staphylococcus aureus and fungal infections) (27, 28). Although our data show that
70 monotherapy can have a marked effect on survival, in practice it is more likely that such an
71 approach would be used as an adjunctive therapy to the limited drugs that are still in clinical use.
72 Such combination therapies are the topic of active investigations.
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73 MATERIALS AND METHODS
74 Organisms. A. baumannii HUMC1 is a MDR clinical strain with susceptibility to colistin (MIC
75 = 2.0 µg/ml) (29). A. baumannii ATCC17978 is a carbapenem sensitive clinical strain
76 (imipenem MIC = 0.25 µg/mL). Carbapenemase-producing Klebsiella pneumoniae (CPKP)-RM
77 and P. aeruginosa are both MDR and obtained from patients at Harbor-UCLA Medical Center.
78 Organisms were grown overnight in tryptic soy broth (TSB) at 37C with shaking. Bacterial
79 cells were passaged for 3 h in TSB, rinsed in phosphate buffered saline (PBS), and the final
80 bacterial counts were determined using MacFarland standard at 600 nm. Susceptibility of the
81 GNB to ARF6 inhibitors was assessed by the CLSI microdilution methods (30).
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83 ARF6 Inhibitors. A high-throughput screen (HTS) was used to identify direct inhibitors of
84 ARF6. The HTS used a fluorometric biochemical assay for monitoring ARF6 activation by
85 measuring exchange of ARF6-bound GDP for the fluorogenic GTP-BODIPY (Life
86 Technologies) (20). Seven distinct structural classes of ARF6 inhibitors as well as 15 singleton
87 hits were identified in our HTS campaign from a library of ~ 50,000 commercially available
88 compounds. We used a structural homology model of ARF6 in complex with its activator ARNO
89 (data not shown) to guide initial medicinal chemistry optimization for the top priority HTS hits.
90 The primary lead candidate series, which is based on pyrazole [1,5-a]pyrimidin-7(4H)-one
91 scaffold, includes the original HTS hit NAV-2729 (20) along with its structural analog of A6-
92 4424 (Fig. S1A) found via substructure searches of commercially available libraries or
93 synthesized as part of a nascent medicinal chemistry program initiated by A6 Pharmaceuticals.
94 A6-5093 is a highly soluble prodrug of A6-4424 designed to enhance in vivo administration.
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95 When A6-5093 is administered in vivo, its lysyl group is rapidly cleaved to produce A6-4424
96 (Fig. S1A and S1E-G)
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98 Animal studies.
f
99 Conditional Arf6 knockout mice. C57BL/6 mice harboring the Arf6 (Arf6 with flanking LoxP
-
-
00 sites (Fig. S4A) and Arf6 alleles have previously been described (16). Endothelial-specific Arf6
/
-
f/f
+/-
01
conditional knockout mice were generated by crossing Arf6 mice with Arf6 ; Tie2-cre mice.
f
02 Excision of the Arf6 allele was verified by isolating lung endothelial cells as previously
03 described (31) and performing a Western blot to assess ARF6 levels using anti-ARF6 antibody
04 from Cell Signaling Technology (#3546) (Fig. S4B).
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06 Infection with GNB. Male ICR mice (20-23 g) were obtained from Envigo. Mice (ICR or Arf6
07 knockout mice) were immunosuppressed using 200 mg/kg cyclophosphamide (intraperitoneal
08 [IP] administration) and 250 mg/kg cortisone acetate (subcutaneous administration) on Days -2
09 and +3 relative to infection. The current regimen results in pancytopenia for at least 9 days from
10 the first administered dose (23). Mice were infected with aerosolized A. baumannii HUMC1
7
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11 (18), or intratracheal instillation of CPKP (1.2 x 10 cells) or P. aeruginosa (2.4 x 10 cells).
12 Treatment with ARF6 inhibitor (30 mg/kg once daily of active moiety given IP) started 3 h post
13 infection. Infected mice treated with vehicle or those treated with 2.5 mg/kg of colistin given
14 twice daily (bid) acted as controls. All treatments continued daily for seven days. The primary
15 endpoints were time to moribundity. In a separate set of experiments, secondary endpoints using
16 organs harvested on Day +4 relative to infection included tissue bacterial burden by quantitative
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17 culturing, inflammatory response, and histopathological examination using hematoxylin and
18 eosin or Gram stain.
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20 Cytometric Bead Array (BD Biosciences) per the manufacturer’s instructions.
21 Mouse organ permeability was determined by injecting Evans blue dye (EBD) (Sigma) 3
The inflammatory cytokine response (IL-1β, IL-6, and TNF-α) was determined using
22 days after infection, harvesting organs, and processing them as previously described (12, 13).
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24 Pharmacokinetic (PK) studies.
25 Mouse. ARF6 inhibitor (NAV-2729 or A6-5093) was administered to male C57BL/6 mice by IP
26 injection. Mice were euthanized at various times after drug administration and blood was
27 collected. Plasma was analyzed for presence of ARF6 inhibitor by a liquid chromatography/mass
28 spectrometry (LC/MS) method. PK data were analyzed with Phoenix WinNonlin (Certara, Inc.)
29 Rat. ARF6 inhibitor (A6-5093) was administered to male Sprague-Dawley rats by intravenous
30 (IV) injection given over approximately 5 sec. Blood was harvested from indwelling arterial
31 catheters at up to 10 time points over a 48-hr period after drug administration. Plasma was
32 analyzed for presence of ARF6 inhibitor by an LC/MS method. PK data were analyzed with
33 Phoenix WinNonlin (Certara, Inc.)
34
35 Endothelial cells. Human umbilical vein endothelial cells (HUVECs) were collected by the
36 method of Jaffe et al. (32) and passaged twice prior to using in assays. HUVEC propagation was
o
37 at 37 C in 5% CO2 (33, 34). Reagents were tested for endotoxin using a chromogenic limulus
38 amebocyte lysate assay (Charles River Laboratories), and the endotoxin concentrations were
39 <0.01 IU/ml.
16

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41 Transwell permeability assay. HUVECs were seeded on 0.4 μm transwell inserts (Corning)
42 coated with fibronectin (10-15 µg/ml in PBS) and fitted in 24-well plates (Costar). HUVECs
43 were grown to confluency in M-199 medium without phenol red and then challenged with 10
7
o
44 bacterial cells for 3 h at 37 C. Following incubation, 3 µl of 50 mg/mL FITC-dextran-10K
45 (Sigma) was added to the top chamber and the amount of vascular leak was determined 1 h later
46 by quantifying the concentration of the dye in the bottom chamber using florescence microplate
47 reader at 488 nm.
48
To investigate the effect of stimuli or inhibitors on HUVECs permeability, the assay was
49 repeated with or without 50 µM ARF6 inhibitors (A6 Pharmaceuticals), 50 µg/ml anti-TLR4
50 antibody or isotype control antibody (Biolegend), or 100 ng/ml E. coli LPS. To devoid bacterial
51 supernatant of any shedded LPS, supernatants were treated with polymyxin B agarose beads.
52
53 ARF6 pulldown assays. HUVECs were treated with A. baumannii HUMC1 with or without 50
54 µM ARF6 inhibitor for 4 h. The cells were washed with ice-cold PBS and lysed with ice-cold
55 lysis buffer supplemented with protease and phosphatase inhibitor (Thermo Scientific, # 26186).
56 Immunoprecipitation (IP) was performed with GST-GGA3-conjugated agarose beads (Cell
57 Biolabs) (13). Protein level of GTP-bound ARF6 and total ARF6 in cell lysates was detected by
58 Western blot with an anti-ARF6 antibody (1:1000 in 5% milk) (Sigma).
59
60 HUVEC gene knockdown. HUVECs were trypsinized and resuspended in growth medium with
61 30 nM of siRNAs targeting MyD88 (SI00300909), ARNO (SI00061299), and ARF6
62 (SI02757286) using HiPerFect transfection reagent (Qiagen) (13) and incubated for 2 days at
17

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63 37 C. The transfection process was repeated once more. All-Stars negative control siRNA
64 (Qiagen) was used as a control. RNA from transfected HUVEC was isolated using RNeasy Plus
65 Mini Kit (Qiagen) after incubation for 4 h with or without A. baumannii HUMC1. cDNA was
66 synthesized using RETROscript Reverse Transcription kit (Thermo Fisher Scientific). qPCR
67 reaction was prepared with Taqman Gene expression assay probes (Thermo Fisher Scientific)
68 and TaqMan gene expression Master Mix (Thermo Fisher Scientific). The qPCR was carried out
69 with a thermal-cycling program as follows: initial denaturing step for 10 min at 95°C, followed
70 by 40 cycles of denaturing at 95°C for 15 s, and annealing/elongation at 60°C for 1 min. Human
71 18s rRNA gene probe (Hs00917508_m1) was used as a reference control. Real-time Probes of
72 targeted genes were Human ARF6 (Hs01922781_g1), MyD88 (Hs00851874_g1), and ARNO
73 (Hs00244669_m1). The comparative Ct method (ΔΔCT) was used for data analysis.
74
75 VE-cadherin expression by confocal microscopy. HUVECs were seeded on coverslips that had
76 been pre-coated with fibronectin in 24-well plates. HUVECs were incubated with A. baumannii
77 HUMC1 for 4 h with or without 50 µM of ARF6 inhibitor, and then washed twice with PBS,
78 fixed with 4% formaldehyde for 10 min. The cells were washed 4 times with PBS, and then
79 blocked with 300 µl 50% human AB-serum (Sigma) for 10 min at room temperature. Mouse
80 monoclonal VE-Cadherin antibody (BD Biosciences, # 555661) was added to the HUVECs (2.5
81 µg/ml) and held overnight at 4°C. Next, HUVECs were washed with PBS, counter stained with 2
82 mg/ml Alexa Fluor® 594 F(ab’)2 goat anti-mouse IgG (Thermo Fisher Scientific, 1:300 in 2%
83 human serum-PBS) for 1 h at room temperature. The cells were stained with 1 µg/ml Hoeschst
84 33342 (ThermoFisher) in PBS for 10 min. HUVECs were washed twice with distilled water, and
85 wet mounted on glass slides, before examining by confocal microscopy.
18

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87 VE-Cadherin quantification by flow cytometry. HUVECs were seeded in 6-well plate
88 (Costar) pre-coated with fibronectin. HUVECs were incubated with A. baumannii HUMC1 for 4
89 h with or without 50 µM of ARF6 inhibitor, and then washed twice with PBS. HUVECs were
90 detached from the plate with cellStripper dissociation reagent (ThermoFisher) that had been
91 prewarmed at 37C for 10 min. M199 medium containing 20% FBS was added to each well and
92 the cells collected. HUVECs were centrifuged, washed with PBS, and then fixed with 0.5 ml of
93 4% formaldehyde at room temperature for 15 min. The fixed cells were washed with PBS,
94 blocked with 50% human AB-serum, and then incubated with 2.5 µg/ml mouse monoclonal VE-
95 cadherin antibody (BD Biosciences, # 555661) overnight at 4°C. The next day, cells were
96 washed with PBS and then incubated with Alexa Fluor® 488F(ab’)2 goat anti-mouse IgG
97 secondary (ThermoFisher) (1:300 in 2% human serum-PBS from a stock of 2 mg/ml) for 1 h at
o
98 4 C. Cells were washed 3 times with 2% serum-PBS, and the cells were transferred to FACS
99 tubes. Fluorescence of VE-cadherin was quantified by using a FACSCalibur (Becton Dickinson)
4
00 instrument equipped with an argon laser emitting at 488 nm. Fluorescence data (10 events) were
01 collected with logarithmic amplifiers and % fluorescence was calculated using the CellQuest
02 software.
03
04 Ethics Statement
05 All procedures involving mice were approved by the IACUC of the Lundquist Institute for
06 Biomedical Innovations at Harbor-UCLA Medical Center (Protocol number 30449), according to
07 the NIH guidelines for animal housing and care. Moribund mice according to detailed and well-
19

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08 characterized criteria were euthanized by pentobarbital overdose, followed by cervical
09 dislocation.
10
11 Statistical analysis. Each in vitro experiment was carried out in triplicate and the experiment
12 repeated at least twice. Survival was compared by the nonparametric log-rank. Categorical
13 variables were compared with the Mann Whitney U test for unpaired comparisons. A P value <
14 0.05 was considered significant.
15
20

416 Acknowledgments
4
17
4
18 This work was supported by the National Institutes of Health [R22-R33 AI119339 and R01
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19 AI063503 to ASI; R01 HL077671 and R01 EY025342 to WZ; R01 HL130541, R01 AR064788,
20 and R01 CA202778 to SJO; 1R43 HL127886-01, 2R44 HL127886-02A1, and 5R44 HL127886-
21 03 to Navigen, Inc], and by UCLA CTSI grant UL1TR000124. The funders had no role in the
22 study design, data collection, decision to publish or preparation of the manuscript.
23
We thank Ronell Lopez and Francisco Bautista for technical assistance with genotyping
24 mice and helping with animal studies and Diana Lim for graphical assistance. We also thank Lise
25 Sorensen and Yi Huang for technical assistance and Jacob Winter, Guy Zimmerman, and
26 Michael Matthay for comments regarding the manuscript.
27
This work was presented in part at the ASM Microbe meeting held between June 16-20,
28 2016, Boston, MA and at the 2md International Symposium on Alternative to Antibiotics (ATA)
29 held between December 12-15, 2016. Paris, France.
30
31 Conflict of Interest
32 I have read the journal’s policy and the authors of this manuscript have the following competing
33 interests: A.L.M., Z.T., E.K.T. and A.B. are employees of A6 Pharmaceuticals, LLC, a company
34 that is developing ARF6 inhibitors for treatment of a variety of conditions. A6 Pharmaceuticals,
35 is a wholly owned subsidiary of Navigen, Inc. D.Y.L. is co-founder of Navigen, and is currently
36 employed by Merck & Co., Inc. S.J.O. and A.S.I. have stock options in Navigen.
37 Navigen, a biotechnology company owned in part by the University of Utah Research
38 Foundation, has a license from the University of Utah to develop the ARF6 inhibitors.
39 The rest of the authors declare no conflict of interest.
21

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59 Figures
60 Figure 1. Endothelial cell Arf6 deletion increases survival rates and reduces disease severity
f/+
61 due to MDR A. baumannii pneumonia. (A) Survival of Arf6 mice (n=22) or endothelial cell
f/-
62 ARF6 null mice (Arf6 ; Tie2-cre) (n=17) with A. baumannii pneumonia. (B) Lung bacterial
63 burden (n=12/arm); (C) lung permeability and (D) histopathological examination of lungs with
f/+
64 H&E harvested from Arf6 or endothelial cell ARF6 null mice 4 days post infection with A.
f/+
65 baumannii via inhalation. Lungs from uninfected Arf6 is included as a control. Black and
66 white arrows in (D) denote tissue edema and hemorrhage in wild-type mice, respectively. Bars
67 are 20 µM. Data in (B) and (C) are presented as the median + interquartile ranges.
68
69 Figure 2. Pharmacologic inhibition of ARF6 increases survival rates and reduces disease
70 severity due to MDR GNB infection. ARF6 inhibitors (A6-5093, NAV-2729, and A6-4424)
71 increase survival (A, n=20 mice for all groups except A6-5093 and colistin which had 10 and 19,
72 respectively) and reduce lung bacterial burden (B) of immunosuppressed mice (200 mg/kg IP
73 cyclophosphamide and 250 mg/kg subcutaneous cortisone acetate given on Days -2 and +3
74 relative to infection) with MDR A. baumannii pneumonia. Lungs were harvested on Day +4,
75 relative to infection. *P<0.05 vs. vehicle-treated mice and **P<0.02 vs. all others in (A) and
#
76
P<0.05 vs. vehicle-treated mice, ***P<0.03 vs. all others in (B). ARF6 inhibitors increase
77 survival rate of immunosuppressed mice infected with MDR CPKP (n=10/group) (C), or MDR
78 P. aeruginosa (n=20/group) (D). Log Rank test for all analyses of survival and Wilcoxon Rank
79 sum test for lung bacterial burden.
5
80
5
81 Figure 3. ARF6 inhibition reduces organ permeability and disease severity without
582 affecting the inflammatory response to A. baumannii pneumonia. (A) ARF6 inhibitors
26

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83 (NAV-2729 and A6-5093) decreased permeability in the lungs and kidneys of A. baumannii
‡
84 HUMC1-infected mice. P<0.05 vs. other treatments. (B) NAV-2729 (30 mg/kg) reduced
85 severity of A. baumannii infection as shown by histopathological examination of lungs with
86 H&E stain and spleen with Gram stain. * denotes necrobiosis in spleen harvested from vehicle-
87 treated mice, and to a lesser extent colistin-treated mice, but not from mice treated with NAV-
88 2729. Bars represent 100 µM. (C) IL-6 level in lungs and plasma of A. baumannii HUMC1-
§
89 infected mice and treated with vehicle control, NAV-2729, or colistin. P<0.02 vs. uninfected
90 mice. Organs of neutropenic mice were harvested on day +3 for (A) and day +4 for (B) and (C),
91 relative to infection. Data in (A) and (C) are presented as the median + interquartile ranges.
92
93 Figure 4. A. baumannii-mediated HUVEC permeability is induced by LPS-TLR4 signaling
94 through ARF6 B. (A) A. baumannii HUMC1 (virulent MDR) cells and supernatants induce
95 HUVEC permeability to a similar level as E. coli LPS. Removing LPS by polymyxin B blocks
96 this induction. ATCC 17978 (avirulent, drug sensitive) cells and supernatants do not induce
97 permeability. *P <0.005 vs. HUMC1, HUMC1 supernatant, or E. coli LPS. (B) A permeability
98 assay was conducted with HUMC1 or its supernatant in the presence of 50 µg/ml of anti-TLR4
99 or isotype-matched control antibodies. *P <0.001 vs. isotype-matched antibody. (C), ARF6-GTP
00 pulldown assays show that A. baumannii HUMC1 induces ARF6 activation and NAV-2729
01 blocks it. Quantification of the ARF6-GTP/total ARF6 ratio by densitometer (3 independent
02 experiments) shows A. baumannii induces endothelial cell ARF6 activation by 2-fold, while
03 NAV-2729 inhibits this activation. *P <0.05 vs. all other comparators. (D) A HUVEC
04 permeability assay was conducted with HUMC1 in the presence of 50 µM ARF6 inhibitors
05 (NAV-2729 or A6-4424). *P <0.0015 vs. HUMC1 cells.
27

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06 Figure 5. A. baumannii-mediated HUVEC permeability is induced through the
07 MyD88/ARNO/ARF6 pathway. (A) Successful down regulation of gene expression (qRT-
08 PCR) using siRNA constructs targeting ARF6, ARNO, or MyD88, in HUVECs. *P <0.001 vs.
09 untransfected HUVECs (2 experiments). (B) (C) ARF6 expression as determined by qRT-PCR
10 and Western blot (B) and A. baumannii-mediated permeability using FITC-dextran (C) of
11 HUVECs transfected with siRNA constructs targeting MyD88, ARNO, ARF6 or scrambled
12 sequence (control). *P<0.008 vs. control siRNA + A. baumannii (B), and *P <0.002 vs. control
13 siRNA + A. baumannii (C).
14
15 Figure 6. A. baumannii compromises HUVEC integrity by promoting internalization of
16 VE-cadherin. (A) Confocal images of HUVECs incubated with A. baumannii HUMC1 (AB)
17 with or without 50 µM NAV-2729 and stained with anti-VE cadherin antibody showing decrease
18 in surface localization of VE-cadherin (red stain) at cell-cell junctions in the absence of NAV-
19 2729 (cyan arrows) and restoration of junctional VE-cadherin with NAV-2729 to levels seen
20 with uninfected HUVECs (white arrows). Cell nucleus is stained blue. Bars are 50 µM. (B)
21 representative flow cytometry staining of surface expression VE cadherin and (C) its
22 quantification from three independent experiments.
6
23
6
24 Figure 7. Model of GNB-induced vascular leak and the role of ARF6 inhibitor in
625 preserving vascular integrity and increasing survival in bacterial infections. (A) In a normal
626 host, the vasculature is held intact by VE-cadherin localized to the cell-cell junctions. (B) In
627 bacterial sepsis (e.g. A. baumannii, AB), LPS-induced endotoxemia triggers a robust host
628 inflammatory response by TLR4-mediated activation of MyD88/NF-κB required for clearing the
629 infection. (C) Bacterial LPS also triggers ARF6 activation via MyD88/ARNO which leads to
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630 intercellular recruitment of VE-cadherin resulting in increased vascular leak, tissue edema, organ
631 failure and ultimately death. (D) ARF6 inhibitors prevent ARF6 activation, which reduces VE-
632 cadherin internalization resulting in the preservation of vascular integrity without affecting the
633 inflammatory immune response.
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