Chitin Derived Small Molecule AVR-48 Reprograms the Resting Macrophages to an Intermediate Phenotype and Decrease Pseudomonas aeruginosa Mouse Lung Infection

: AVR-48 is a structural derivative of chitin previously shown by our laboratory to signiﬁ-cantly decrease lung injury parameters in LPS, hyperoxia and sepsis-induced rodent models. The current study objectives are to determine the cellular mechanism of action and demonstrate efﬁcacy in a mouse bacterial lung infection model. For in vitro receptor binding and macrophage polarization studies, C57Bl/6J mouse derived spleens and human peripheral blood mononuclear cells (hPBMCs) were treated with AVR-48 ± LPS or biotin conjugated AVR-48. Different macrophage types were determined using ﬂow cytometry and secreted cytokines were measured using ELISA. In vivo, a CD-1 mouse Pseudomonas aeruginosa lung infection was treated with AVR-48, assessing bacterial colony forming unit (CFU), IL-10 and IL-17A levels in lung and blood samples. AVR-48 binds to both the toll-like receptor 4 (TLR4) and the CD163 receptor on mouse monocytes. In hPBMCs, frequency of intermediate macrophages increased upon AVR-48 treatment for 72 h. Increased bacterial phagocytosis/intracellular killing were observed in THP-1 cells and reduction in CFU in CD-1 mouse lungs. Binding of AVR-48 to both TLR4 and CD163 receptors bring the macrophages to an intermediary stage, resulting in increased phagocytosis and decreased inﬂammation, altogether providing an optimal immune balance for treating lung injury and infection.


Introduction
Chitin and chitosan are high molecular weight oligosaccharides with diverse biological activities, having anti-microbial, anti-inflammatory, anti-oxidant, anti-tumorigenic, immunostimulating, and calcium-and iron-absorption-acceleration properties [1]. Chitohexaose, a hexaoligomeric chitosan, binds to the Toll-Like Receptor (TLR4). TLR4 is a pattern recognition receptor (PRR) through which bacterial lipopolysaccharide (LPS) induces an innate immune response. Our previous studies suggested that [2] chitohexaose, after binding with TLR4, modulates macrophages to a non-inflammatory M2 phenotype and downregulates LPS induced inflammatory mediators such as IL-1β, TNF-α and IL-6. This mechanism of action (MoA) for chitohexaose was confirmed in a TLR4 mutant C3H/Hej mice model by Panda et al. [2]. The proprietary first generation novel chitohexaose analog, AVR-25, demonstrated marked efficacy in decreasing lung injury and inflammation in a hyperoxia-induced acute lung injury model in adult mice [3]. In addition, we demonstrated Immuno 2022, 2 652 that AVR-25 prevents lung injury in an hyperoxia-induced bronchopulmonary dysplasia (BPD) murine pup model [3,4]. Furthermore, AVR-25 was very effective in protecting mice against cecal ligation and puncture (CLP) induced polymicrobial infection and sepsis [5]. To allow an efficient manufacturing and formulation process, we further optimized the chemical structure of AVR-25 using a medicinal chemistry structure activity study to obtain a series of lower molecular weight compounds [6]. Recently we have reported that one of the optimized compound, AVR-48, significantly decreases lung injury, pulmonary edema, permeability and inflammation in both hyperoxia and LPS-induced Acute Lung Injury (ALI) and CLP-induced sepsis in murine models [3]. AVR-48 also was very effective in preventing hyperoxia-induced lung injury and pulmonary hypertension in an experimental bronchopulmonary dysplasia (BPD) model in new-born murine pups [7].
In this study, we investigated the possible immunomodulating MoA of AVR-48 by addressing the following key issues: (1) does AVR-48 have selective binding affinity to surface receptors on monocytes and/or macrophages over other mononuclear immune cells including T cells and B cells, (2) does AVR-48 have binding affinity to any other surface receptors on monocytes and macrophages besides TLR4, (3) does binding of AVR-48 to TLR4 agonize or antagonize the downstream signaling cascade, (4) does AVR-48 treatment to human peripheral blood mononuclear cells (hPBMCs) promote differentiation into different sets of macrophages (M1, M2 or an intermediate (M int ) phenotype) and whether these macrophages can phagocytose pathogenic bacteria, (5) does AVR-48 treatment reduce significant bacterial loads and inflammation in lung tissue following infection?
To address these questions, we used both murine splenic monocytes (MSMs) and hPBMCs for determining the binding efficacy of AVR-48 in mononuclear cell receptors, macrophage polarization and cytokine release studies. We used THP-1 human monocyte cell lines to test AVR-48 induced bacterial membrane phagocytosis and intracellular killing potential. Lastly, we demonstrated the in vivo antibacterial and anti-inflammatory activity using a mouse model of Pseudomonas aeruginosa lung infection.

Animals
Both female CD-1 and male C57BL/6J mice were purchased from Taconic (Rensselaer, NY 12144, USA) and were maintained in a breeding colony at the University of North Texas Health Science Center (Fort Worth, TX, USA) Animal Care Facilities with Animal Welfare Assurance No. D16-00417 (A3711-01). Animal procedures were performed in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of UNTHSC, TX (Protocol no. 2020-0002).

Cells and Cell Lines
hPBMCs were directly purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA, catalog no. 690PB-100A) and were used as per manufacturer guidelines. THP-1 human leukemic monocyte cells were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA, catalog no. TIB-202) and authenticated prior to receipt for their identity and validity. For THP-1 cells from all sources, the original stock was expanded and aliquoted into individual tubes prior to freezing in liquid nitrogen. The purpose was to avoid contamination of the stock and to maintain access to as much of the validated original sample as possible. Limiting the use of the products of each aliquot to 2-3 passages ensured maintenance of the integrity of the cell lines being used.

Chemicals and Reagents
The synthesis and structural characterization of AVR-48 and biotinylated AVR-48 (BTAVR-48) was conducted in the laboratory of AyuVis Research Inc., following the inhouse procedures [6]. Endotoxin-free phosphate-buffered saline (PBS), phorbol myristyl acetate (PMA), meropenem, imipenem, ciprofloxacin and colistin were purchased from Sigma-Aldrich Inc., St. Louis, MO, USA. Ultrapure LPS derived from Salmonella minnesota was purchased from InvivoGen, San Diego, CA, USA (catalog no. tlrl-smlps). Formulation of AVR-48 for in vitro and in vivo studies was done in saline.
For the mouse studies, AVR-48 was reconstituted in 0.9% sterile normal saline to prepare a final dose concentration of 10 mg/kg as a colorless solution, filtered with 0.2 µM syringe filter and injected IP (30 µL). For cellular assays, a 10 mM stock solution of AVR-48 was prepared in saline and further diluted with culture media to attain desired concentrations.

Cell Viability Assay
hPBMCs (1 × 10 5 ) were plated in a 96 well plate and treated with either AVR-48 (0.1, 1.0, 10, 100 and 1000 µM), or BTAVR-48 (1, 10 and 100 µM) and incubated for 48 h. The cells were treated with MTT and resolving reagents (Promega, Madison, WI, USA), incubated for 2 h and read at 450 nm absorbance following manufacturer's instruction. The percentage of viable cells was calculated from the change in absorbance of the AVR-48 treated cells as compared to untreated control cells. Studies were performed in triplicate.

IL-10 and IL-17A Detection in Lung Homogenates and Serum
Upon sacrifice, mice lung tissues were harvested and homogenized in PBS and stored at −80 • C until analysis. Serum samples were collected from anti-coagulated whole blood and stored at −80 • C until analysis. The concentration of IL-10 and IL-17A in total lung homogenates and serum were determined by sandwich ELISA method. All procedures were performed as described by the manufacturer. IL-10 and IL-17A concentrations were determined according to standard curve generated by reference concentration of cytokine at a wavelength of 450 nm detected by colorimetric plate reader (Biotek Instruments Inc. Winooski, VT, USA). ELISA antibody set and recombinant cytokine for standard were purchased from eBiosicence (eBiosciences, San Diego, CA, USA).

Combination MIC Assay
The individual MIC 90 of the antibiotics meropenem, ciprofloxacin, colistin and compound AVR-48 was determined first using a broth dilution assay for Pseudomonas aeruginosa (ATCC-10145), Acinetobacter baumanni (ATCC-19606) and methicillin resistant Staphylococcus aureus (ATCC-BAA-1760) bacteria. To determine the synergy and the combination MIC of AVR-48 and different standard of care (SoC) antibiotic, a checkerboard design was used as described earlier [9]. In a 96 well plate, AVR-48 was added horizontally diluted two-fold from left to right where antibiotics were added from top to bottom at their MIC, 1/2 of MIC and at 1/4 of MIC concentrations. Then 5 µL of bacteria was added to each well and incubated for 6 h; the plate was read at 600 nM. The MIC 90 was calculated from the change absorbance. Fractional inhibitory concentrations (FICs) were calculated by use of the following formula: FIC (X + Y) = [MIC of compound X in combination with Y]/[MIC of X alone]. The fractional inhibitory index (∑FIC) was calculated as FIC of compound X + FIC of compound Y to evaluate interaction profiles. ∑FICs of ≤0.5 designates synergistic activity, ∑FICs of ≥4.0 indicates antagonism, and values in between correspond to additive, as outlined in previous work on antibacterial combination studies [10,11].

Pseudomonas aeruginosa Mouse Lung Infection
Briefly, groups of female CD-1 (n = 3-4/group) were lightly anesthetized and subjected to an inoculum of 1 × 10 6 CFUs of P. aeruginosa (ATCC BAA-2108) in two 20 µL droplets over the nares. Following an 8-h incubation period, groups of mice were intravenously administered with AVR-48 (10 mg/kg/dose, q12 h) alone, meropenem alone (1.5 mg/kg, q12 h) or in combination for 3 days. The compound was formulated in saline and injected using IV tail vein injection. One of the remaining groups was sham-treated with vehicle when the AVR-48 was administered, and the other two remaining groups remained untreated. Mice were euthanized at 72 h after infection, and blood and lungs were harvested from each animal. Total CFUs were determined from each blood and lung homogenates and processed for cytokine by ELISA.

Quantification of Bacterial Load in Mouse Lung and Blood Samples
Bacterial load in lung and blood were evaluated after infection. Upon sacrifice, lung tissues were harvested and homogenized in PBS. Ten-fold serial dilution of lung homogenates were plated onto blood agar plates and incubated overnight at 37 • C with 5% CO 2 . Colonies on plates were enumerated, and the results were expressed as log 10 CFU/mL per organ. Similarly, heparinized blood samples were serial diluted at 1:10 concentrations and plated onto blood agar plates. Colonies on plates were enumerated after overnight incubation at 37 • C with 5% CO 2 . The results were expressed as log 10 CFU per mL of homogenized organ.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism Version 8.0 (GraphPad Software, San Diego, CA, USA). For multi-experimental group analysis, data were subjected to one-way ANOVA (analysis of variance) followed by post hoc tests (Dunnett's multiple comparison test, Newman-Keuls and Bonfferoni) for group differences. Homogeneous data were analyzed using the ANOVA, and the significance of intergroup differences between the control and test item-treated groups was analyzed using Dunnett's test. Heterogeneous data was analyzed using Kruskal-Wallis test and the significance of intergroup differences between the control and test item-treated groups were assessed using a nonparametric Dunnett's test. All data are reported as ±SEM. A significance level of p < 0.05 at 95% confidence intervals was considered statistically significant for all the experiments reported in this study. All the experiments are repeated two or three times with 2-4 technical replicates, and the data were pooled together and averaged for statistical analysis.

AVR-48 and Biotin Conjugated AVR-48 Do Not Exhibit Cytotoxicity to hPBMCs In Vitro
First, the effect of AVR-48 and biotin conjugated AVR-48 on hPBMCs in vitro and cell viability was analyzed. Both of the molecules have no cytotoxicity to hPBMCs upon treatment for 48 h. The frequency of live cells was comparable between the control, AVR-48 ( Figure 1A,C) or biotin conjugated AVR-48 (BTAVR-48) treatment groups ( Figure 1B,D) when incubated using low-to-high concentrations. We have observed that upon AVR-48 (0.1 µM, 1 µM and 10 µM) treatment, there is increased cell proliferation as compared to control cells. The plausible reason might be differentiation of monocytes into macrophages at 48 h which have a survival benefit to expand/proliferate as compared to the monocytes. Further investigation is warranted to decipher the exact mechanism.
cell viability was analyzed. Both of the molecules have no cytotoxicity to hPBMCs upon treatment for 48 h. The frequency of live cells was comparable between the control, AVR-48 ( Figure 1A,C) or biotin conjugated AVR-48 (BTAVR-48) treatment groups (Figure 1B,D) when incubated using low-to-high concentrations. We have observed that upon AVR-48 (0.1 µM, 1 µM and 10 µM) treatment, there is increased cell proliferation as compared to control cells. The plausible reason might be differentiation of monocytes into macrophages at 48 h which have a survival benefit to expand/proliferate as compared to the monocytes. Further investigation is warranted to decipher the exact mechanism.

AVR-48 Binds to Both TLR4 and CD163 Receptors in Primary Monocytes
In order to identify the on-target receptors for AVR-48 in the murine splenocytes, we performed a competitive binding experiment using antibodies to putative target and AVR 48. Binding of anti-TLR4 and anti-CD163 antibody to the surface of murine splenic cells was inhibited by BTAVR-48 in a dose-dependent manner ( Figure 2B-E). The maximum binding efficacy (75-78%) was observed at 100 µM concentration for both receptors, and the IC 50 for BTAVR-48 was calculated to be 21.6 µM for TLR4 and 11.6 µM for the CD163 receptor from the dose response curve. We did not observe any binding affinity of AVR-48 for either TLR2 or CD206 receptors (Supplemental Figure S1).
we performed a competitive binding experiment using antibodies to putative target and AVR 48. Binding of anti-TLR4 and anti-CD163 antibody to the surface of murine splenic cells was inhibited by BTAVR-48 in a dose-dependent manner ( Figure 2B-E). The maximum binding efficacy (75-78%) was observed at 100 µM concentration for both receptors, and the IC50 for BTAVR-48 was calculated to be 21.6 µM for TLR4 and 11.6 µM for the CD163 receptor from the dose response curve. We did not observe any binding affinity of AVR-48 for either TLR2 or CD206 receptors (Supplemental Figure S1).

AVR-48 Treatment Polarizes Mouse Monocytes to Macrophages and Shifts the Monocyte Populations More to a Resident Phenotype
Resident macrophages exert increased anti-inflammatory and phagocytic activities desired for resolution of inflammation, clearance of bacteria and tissue repair as compared to inflammatory macrophages [12]. In order to understand the mechanism behind our previously reported anti-sepsis and anti-inflammatory activity of AVR-48 in mouse models [3], we tested the effect of AVR-48 on macrophage polarization. Murine glass adherent splenic cells (considered as monocytes) [13] were stimulated with different doses of AVR-48 for 72 h and were analyzed for expression of different monocyte/macrophage markers. We observed that the expression of the macrophage-specific markers MHC-II and CD11b ( Figure 3B,C) were increased after AVR-48 treatment in a dose-dependent manner. Further staining with macrophage-specific marker F4/80 demonstrated an increase in macrophage population (F4/80 + ) after treatment with either 10 µM or 100 µM concentrations of AVR-48 as shown in Figure 3D. desired for resolution of inflammation, clearance of bacteria and tissue repair as compared to inflammatory macrophages [12]. In order to understand the mechanism behind our previously reported anti-sepsis and anti-inflammatory activity of AVR-48 in mouse models [3], we tested the effect of AVR-48 on macrophage polarization. Murine glass adherent splenic cells (considered as monocytes) [13] were stimulated with different doses of AVR-48 for 72 h and were analyzed for expression of different monocyte/macrophage markers. We observed that the expression of the macrophage-specific markers MHC-II and CD11b ( Figure 3B,C) were increased after AVR-48 treatment in a dose-dependent manner. Further staining with macrophage-specific marker F4/80 demonstrated an increase in macrophage population (F4/80 + ) after treatment with either 10 µM or 100 µM concentrations of AVR-48 as shown in Figure 3D. As described by Swirski et al. [13], in mice Ly6C hi cells are considered inflammatory monocytes whereas Ly6C low cells are resident and anti-inflammatory monocytes. A reciprocal increase of the resident cell population after treatment of splenocytes with AVR-48 for 72 h was observed ( Figure 4B). Additionally, we confirmed that AVR-48's binding As described by Swirski et al. [13], in mice Ly6C hi cells are considered inflammatory monocytes whereas Ly6C low cells are resident and anti-inflammatory monocytes. A reciprocal increase of the resident cell population after treatment of splenocytes with AVR-48 for 72 h was observed ( Figure 4B). Additionally, we confirmed that AVR-48's binding is specific to monocytes/macrophages. Binding of BTAVR-48 to splenic monocytes/macrophages showed a dose-dependent binding to monocytes/macrophages as determined by increased expression of MHCII+ and Ly6C + cells where there was no binding to T cells or B cells (CD3 + , CD19 + ) observed with AVR-48 (Supplemental Figure S2A-C).
is specific to monocytes/macrophages. Binding of BTAVR-48 to splenic monocytes/macrophages showed a dose-dependent binding to monocytes/macrophages as determined by increased expression of MHCII+ and Ly6C + cells where there was no binding to T cells or B cells (CD3 + , CD19 + ) observed with AVR-48 (Supplemental Figure S2A-C).  Figure  S4). The intermediate macrophages positive for HLA-DR are antigen presenting cells (APC) and anticipated to be more phagocytic and pro-inflammatory in nature to kill microbes [14,15]. Macrophages positive for CD206 are both phagocytic and antiinflammatory in nature [16].  Figure S4). The intermediate macrophages positive for HLA-DR are antigen presenting cells (APC) and anticipated to be more phagocytic and pro-inflammatory in nature to kill microbes [14,15]. Macrophages positive for CD206 are both phagocytic and anti-inflammatory in nature [16].

Effect of LPS and AVR-48 Treatment on Percentage of Macrophages in hPBMCs
In the combination study of AVR-48 with LPS in hPBMCs, we observed that the M int population (HLA-DR + CD206 + ) is significantly higher in both LPS-treated groups and LPS+AVR-48 treated groups ( Figure 6B). No significant changes were observed with the M2 (HLA-DR -CD206 + ) macrophage populations between all groups. The overall percentage of HLA-DR + CD206macrophages noted here as M1 macrophages was very small (0.5-1.5% of the parent cells), and we observed no significant change in their numbers ( Figure 6B) after LPS treatment. Interestingly, LPS+AVR-48 treatment groups at all concentrations showed a decrease in percentage of M1 macrophages (20-30%, p < 0.05) compared to either control or LPS-treated cells.

Effect of LPS and AVR-48 Treatment on Percentage of Macrophages in hPBMCs
In the combination study of AVR-48 with LPS in hPBMCs, we observed that the Mint population (HLA-DR + CD206 + ) is significantly higher in both LPS-treated groups and LPS+AVR-48 treated groups ( Figure 6B). No significant changes were observed with the M2 (HLA-DR -CD206 + ) macrophage populations between all groups. The overall percentage of HLA-DR + CD206 -macrophages noted here as M1 macrophages was very small (0.5-1.5% of the parent cells), and we observed no significant change in their numbers ( Figure 6B) after LPS treatment. Interestingly, LPS+AVR-48 treatment groups at all concentrations showed a decrease in percentage of M1 macrophages (20-30%, p < 0.05) compared to either control or LPS-treated cells.

Effect of AVR-48 and LPS on Concentration of TNF-α and IL-6 in hPBMCs
Here we measured the concentrations of inflammatory cytokines TNF-α and IL-6 from the cell supernatant after treatment with either AVR-48 or LPS or both at four different timepoints. There were no significant TNF-α or IL-6 concentrations detected at 6 h and 24 h timepoints after treatment with AVR-48 compared to untreated control cells. There was a significantly (p < 0.05) higher (ca. 1.5-fold) concentration of TNF-α found after treatment with 48 h at 1 µM concentration ( Figure 7A) compared to untreated control cells where no significant increase was noticed at either 10 µM or 100 µM. Similarly, no significant change in IL-6 was noticed in 48 h ( Figure 7C). In control cells, at 72 h, very low levels of TNF-α were detected in cell supernatant where IL-6 concentrations were significantly higher by comparison. To our expectation, AVR-48 at all concentrations significantly decreased IL-6 concentrations ( Figure 7C).

Effect of AVR-48 and LPS on Concentration of TNF-α and IL-6 in hPBMCs
Here we measured the concentrations of inflammatory cytokines TNF-α and IL-6 from the cell supernatant after treatment with either AVR-48 or LPS or both at four different timepoints. There were no significant TNF-α or IL-6 concentrations detected at 6 h and 24 h timepoints after treatment with AVR-48 compared to untreated control cells. There was a significantly (p < 0.05) higher (ca. 1.5-fold) concentration of TNF-α found after treatment with 48 h at 1 µM concentration ( Figure 7A) compared to untreated control cells where no significant increase was noticed at either 10 µM or 100 µM. Similarly, no significant change in IL-6 was noticed in 48 h ( Figure 7C). In control cells, at 72 h, very low levels of TNF-α were detected in cell supernatant where IL-6 concentrations were significantly higher by comparison. To our expectation, AVR-48 at all concentrations significantly decreased IL-6 concentrations ( Figure 7C). LPS treatment significantly increased TNF-α at 6 h, 24 h and 48 h ( Figure 7B). While LPS+AVR-48 treatment decreased the TNF-α concentration at a higher dose of AVR-48 (100 µM) after 6 h, AVR-48 was more potent at 24 h at all concentrations (1, 10 and 100 µM). No significant change in TNF-α concentration is observed at 48 h and 72 h with LPS + AVR-48 treatment. With LPS treatment, IL-6 concentrations were found to be significantly higher at early time points of 6 h and 24 h, but no significant change was noted at 48 h and 72 h ( Figure 7D). Though we observed an increase in IL-6 after LPS + AVR-48 (100 µM) treatment at 24 h, after 72 h, IL-6 level was significantly lower compared to control cells ( Figure 7D).

Effect of AVR-48 and LPS on Concentration of IL-10 and sCD163 in hPBMCs
In Figure 2 we showed that AVR-48 binds to the CD163 scavenger receptor in mouse primary spleen cells with an IC 50 of 11.6 µM. Here we show that in hPBMCs, 48 h treatment of AVR-48 (10-100 µM) significantly increases the level of soluble CD163 in the cell supernatant ( Figure 8A). CD163 release is known to stimulate production of anti-inflammatory cytokine IL-10 which moderately increased (1.2-1.5-fold) at 48 h ( Figure 8C). While LPS treatment increased the sCD163 (Figure 8A,B) and IL-10 concentrations at both 48 and 72 h ( Figure 8C,D), a combination of LPS + AVR-48 significantly decreased the LPS-induced increase in both sCD163 and IL-10 concentrations.  Figure 7D). Though we observed an increase in IL-6 after LPS+AVR-48 (100 µ M) treatment at 24 h, after 72 h, IL-6 level was significantly lower compared to control cells ( Figure 7D).

Effect of AVR-48 and LPS on Concentration of IL-10 and sCD163 in hPBMCs
In Figure 2 we showed that AVR-48 binds to the CD163 scavenger receptor in mouse primary spleen cells with an IC50 of 11.6 µ M. Here we show that in hPBMCs, 48 h treatment of AVR-48 (10-100 µ M) significantly increases the level of soluble CD163 in the cell supernatant ( Figure 8A). CD163 release is known to stimulate production of antiinflammatory cytokine IL-10 which moderately increased (1.2-1.5-fold) at 48 h ( Figure  8C). While LPS treatment increased the sCD163 (Figure 8A,B) and IL-10 concentrations at both 48 and 72 h ( Figure 8C,D), a combination of LPS+AVR-48 significantly decreased the LPS-induced increase in both sCD163 and IL-10 concentrations.

AVR-48 Induce Differentiation of THP-1 Human Monocytic Cells into Macrophages with Enhanced Phagocytosis of the Bacteria and Promote Intracellular Killing
In order to determine the phagocytic ability of the intermediate macrophages (M int ) produced after treatment with AVR-48, we next used THP-1 cells for this study [17,18]. We observed an increased number of macrophages (CD11b + ) after 72-h treatment of AVR-48 at 200 µM concentration ( Figure 9A). PMA was used as positive control. Next, when we exposed these macrophages for 30 min to Pseudomonas aeruginosa bacteria tagged for green fluorescein protein (GFP), there was a significant increase in phagocytosis of the bacteria ( Figure 9B) and subsequent intracellular-killing of them ( Figure 9C) as determined by the CFUs measurement within 30-60 min.

AVR-48 Demonstrates Synergy with Standard of Care Antibiotics
We also tested the synergy and additive bactericidal activity of AVR-48 with several of the standard of care (SoC) antibiotics using a serial broth dilution minimum inhibitory concentration (MIC) determination assay following the checkerboard method [9]. The MIC 90 for AVR-48 was found to be >200 µg/mL for all bacteria. Surprisingly, when AVR-48 was combined with SoC broad-spectrum antibiotics-meropenem, colistin, ciprofloxacin-the MIC 90 measurements of the antibiotics were lowered by two-to tenfold (Table 1), enhancing the anti-bacterial activity. In particular, the MIC 90 of meropenem was lowered twofold for P. aeruginosa, MIC 90 of ciprofloxacin was lowered twofold for A. baumannii and MIC 50 of colistin was lowered more than tenfold for MRSA. Fractional inhibitory concentration ∑MIC was found to be <0.5 for the combinations which is a measure of synergy and additive between the two modalities (Supplemental Figure S6).

AVR-48 Induce Differentiation of THP-1 Human Monocytic Cells into Macrophages wit Enhanced Phagocytosis of the Bacteria and Promote Intracellular Killing
In order to determine the phagocytic ability of the intermediate macrophages (M produced after treatment with AVR-48, we next used THP-1 cells for this study [17 We observed an increased number of macrophages (CD11b + ) after 72-h treatmen AVR-48 at 200 µM concentration ( Figure 9A). PMA was used as positive control. N when we exposed these macrophages for 30 min to Pseudomonas aeruginosa bact tagged for green fluorescein protein (GFP), there was a significant increase in phago tosis of the bacteria ( Figure 9B) and subsequent intracellular-killing of them (Figure as determined by the CFUs measurement within 30-60 min.  Recently we published that AVR-48 at 10 mg/kg/dose significantly decreases lung injury, pulmonary edema, lung permeability and inflammatory cytokines in hyperoxia or LPS-induced acute lung injury (ALI) and cecal-ligation-and-puncture-induced (CLP) sepsis mouse models [3]. AVR-48 also was very effective in preventing hyperoxia-induced BPD and pulmonary hypertension in new-born murine pups [7]. Here we have demonstrated that AVR-48 (IV, 10 mg/kg, 2/d for 3 days)-alone and in combination with a standard of care antibiotic, meropenem-decreased bacterial CFUs in the lungs of mice infected with P. aeruginosa in a mouse model ( Figure 10). Infection with P. aeruginosa increased the bacteria CFU significantly, and treatment with either meropenem or AVR-48 were equally effective in lowering the CFU level to roughly one log reduction. The combination AVR-48+meropenem group demonstrated a more robust (ca. 1.5-log-fold, ca. 95%) reduction in CFU, demonstrating excellent synergy and additive effect ( Figure 10A). Also, the inflammatory cytokine IL-17A was found to be lowest in both lung and blood samples from AVR-48 and the AVR-48+meropenem group ( Figure 10B) where the anti-inflammatory cytokine IL-10 concentration was significantly higher ( Figure 10C

Discussion
Low molecular weight chitin and chitosan inhibit sepsis caused by gram-negative bacteria via activation of an alternate immune pathway and possibly produce nonclassical M2 macrophages with anti-inflammatory properties [6,9]. Specifically, we have shown previously that chitohexaose binds to and inhibits TLR4, the primary receptor to which LPS binds and mediates the consequent acute immune response [9]. But at the same time, activation of TLR4 signaling is also essential for survival in acute lung injury induced either by virulent P. aeruginosa type III secretory toxins found in multi-drug resistant (MDR) strains [5]. Thus, it is required to maintain a balance between inflammation and resolution in order to clear the bacterial load, decrease the hyper inflammation and minimize tissue injury. Key to its therapeutic efficacy, the binding of chi-

Discussion
Low molecular weight chitin and chitosan inhibit sepsis caused by gram-negative bacteria via activation of an alternate immune pathway and possibly produce non-classical M2 macrophages with anti-inflammatory properties [6,9]. Specifically, we have shown previously that chitohexaose binds to and inhibits TLR4, the primary receptor to which LPS binds and mediates the consequent acute immune response [9]. But at the same time, activation of TLR4 signaling is also essential for survival in acute lung injury induced either by virulent P. aeruginosa type III secretory toxins found in multi-drug resistant (MDR) strains [5]. Thus, it is required to maintain a balance between inflammation and resolution in order to clear the bacterial load, decrease the hyper inflammation and minimize tissue injury. Key to its therapeutic efficacy, the binding of chitosan/chitohexaose to TLR4 happens in such a way that there is selective activation of the target cell and increase in expression of the important anti-inflammatory cytokine IL-10 [9]. The anti-inflammatory cytokine IL-10 is an important endogenous regulator of several pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and chemokine expression in acute lung inflammation [10].
AVR-48 is an optimized second-generation structural analog and is a derivative of chitohexaose with improved physicochemical properties. Based on our published pharmacokinetic study [7], we demonstrated that the therapeutic activity of AVR-48 is systemic, and immune cells circulating in blood are the possible target cells for the mode of therapeutic action shown by AVR-48. Macrophages are tissue-resident professional phagocytes and antigen-presenting cells (APC), which differentiate from circulating monocytes. They perform important active and regulatory functions in innate as well as adaptive immunity [19]. Primary splenocytes serves as the major pool of lymphocytes and monocytes that further get activated to inflammatory (M1) or non-inflammatory (M2) macrophages depending on exposure to various physiological and pathological stimuli [20]. Macrophages are highly heterogenous cells that can rapidly change their function in response to local microenvironmental signals which can allow conversion of the initial M1/M2 polarization processes; for example, M2-polarized macrophages can convert to the M1-activated status under certain conditions and vice versa. The classically activated M1 macrophages comprise immune effector cells with an acute inflammatory phenotype. These are highly aggressive against bacteria and produce large amounts of lymphokines [21]. The alterna-tively activated, anti-inflammatory M2 macrophages can be separated into at least three subgroups. These subtypes have various different functions, including regulation of immunity, maintenance of tolerance and tissue repair/wound healing. The general protocol of in vitro differentiation of monocytes into M1 or M2 macrophages takes a minimum of 6 days and the use of either granulocyte-macrophage colony-stimulating factor (GM-CSF) or M-CSF and a combination of cytokines/chemokine cocktails. To our utmost surprise, we found that treatment with 100 µM of AVR-48 for 3 days differentiated the primary mouse splenocytes into macrophages (ca. 80%). Swirski et al. [13] reported that mouse spleen cells with Ly6C Hi are considered inflammatory whereas Ly6C low are considered resident and anti-inflammatory. Here we found a higher percentage of resident monocyte populations after AVR-48 treatment and a lower number of inflammatory monocytes. Most importantly, we found that AVR-48 demonstrates selectivity for binding to monocytes and not to other mononuclear immune cells including T cells and B cells. Encouraged by this finding, we conducted further studies to identify what is (are) the putative target receptor(s) to which AVR-48 binds and to agonize/antagonize the receptor(s) to provide the therapeutic activities. We first probed for both TLR4 and TLR2 receptors. Using a biotin tagged AVR-48, we confirmed the binding of AVR-48 to TLR4 and selectivity over TLR2 in mouse spleen cells. What was important at this point was to delineate further to find out whether binding of AVR-48 to TLR4 protein expressed in the surface of monocytes/macrophages leads to activation, inhibition or modulation of the receptor protein. In our published work using a juvenile mouse model of BPD [7], we demonstrated that AVR-48 treatment to both room air (RA) control and hyperoxia (100% O 2 ) mouse pups increased the TLR4 protein concentration in lung tissues after 10 days post last dose of AVR-48 treatment. This increased TLR4 protein concentration coupled with an increase in lung cytokines TNF-α, IL-6, IL-1β and IL-10 supported possible activation of the TLR4 pathway by AVR-48 in naïve animals. However, the concentration of these inflammatory cytokines was significantly lower in both lung and plasma samples obtained from hyperoxia+AVR-48 mouse pups as compared to only hyperoxia groups, indicating a differential MoA by AVR-48 in physiological and pathological scenario which was intriguing.
Our macrophage differentiation study confirmed that there is a fluidic event of macrophage polarization happening after treatment of AVR-48 to hPBMCs. After 48h treatment with either AVR-48 or AVR-48+LPS groups, the monocytes are predominantly differentiating into macrophages (80-90%) positive for HLA-DR antigen, M1 marker CD86 and negative for M2 marker CD206 (Supplementary Figure S4); at 72 h the percentage of intermediate (M int ) macrophages positive for HLA-DR antigen, M1 marker CD86 and M2 marker CD206, dominate (70-80%). The intermediate macrophages positive for HLA-DR are antigen-presenting cells (APC) and anticipated to be more phagocytic and proinflammatory in nature to kill microbes [14,15]. Low-level HLA-DR antigen expression on peripheral blood monocytes correlates with infection [14]. Macrophages positive for CD206 are both phagocytic and anti-inflammatory in nature [16]. The activation of monocyte or resting macrophages (M0) to M1 phenotype at an earlier timepoint can be explained as activation of TLR4 by AVR-48 similar to the known TLR4 agonist LPS. However, it was not very clear why there is an alternate activation and how the percentage of CD206 (M2 phenotype) macrophages are increasing at a later timepoint. In general, macrophage colony stimulating factor (M-CSF) and IL-10 are known to stimulate the macrophages to M2 phenotypes [22]. Here, we did observe an increase in IL-10 concentration in hPBMCs supernatants as well as in the plasma and lung from the mouse model of lung infection.
Additional investigation was made to identify any alternative on-target effect of AVR-48. We hypothesized that AVR-48 might have binding affinity to other receptors specific to M2 macrophages such as scavenger receptor CD163 or mannose receptor CD206 that are expressed abundantly in M2 macrophages. CD163 is a transmembrane scavenger receptor found on the surface of macrophages. Both CD163 and CD206 are expressed by macrophages, but the expression of the receptors is differentially regulated. CD163 expression is increased in response to IL-10 stimulation, while CD206 expression is upregulated by IL-4 and IL-13 [23]. Procherary et al. [24] demonstrated that the activation state of CD163 and CD206 is reversible depending on the microenvironment, suggesting that a given cell may participate sequentially in both the induction and the resolution of inflammation. Cell surface expression of CD163 on alveolar macrophages is reduced in human subjects with asthma [25], BPD [26] and sepsis which suggests that CD163 may participate in the regulation of airway inflammatory responses in the lung. CD163 is released in the circulation in its soluble form, sCD163, via cleavage of the extracellular domain by matrix metalloproteases (MMPs) following oxidative stress [27] or via activation of TLR4 after inflammatory stimuli [28,29], and it is used as a biomarker for increased injury/inflammation. sCD163 protects monocytes from hyperactivation during bacterial infections by dampening the secretion of the proinflammatory cytokines TNF-α, IL-1β, IL-6 and IL-8 [30] and by endocytosing inflammatory neutrophils, excess hemoglobin/haptoglobin complexes responsible for increasing reactive oxygen species (ROS), as well as bacteria/viruses, possess phagocytic activity in clearing both microbes and cell debris. Here, we demonstrated that AVR-48 binds to the CD163 receptor in mouse splenocytes in a dose-dependent manner with an IC 50 of 11.6 µM. CD163 stimulation is known to produce anti-inflammatory cytokine IL-10. We did observe an increase in IL-10 concentration after 48 h of treatment with AVR-48 in hPBMCs which indicated that binding to CD163 receptor resulted in receptor stimulation. IL-10 is also known to negatively regulate production of IL-1β [19,31], an important endogenous regulator of chemokine expression in acute lung inflammation [20]; appears to be protective against the development of BPD [32][33][34][35][36][37], and IL-10-induced microRNA-187 negatively regulates TNF-α, IL-6 and IL-12p40 production in TLR4-stimulated monocytes [38]. The time and dose-dependent IL-10 and sCD163 measurement in plasma at 48 and 72 h after treatment with AVR-48 or LPS or both to PBMC showed that there is a dose-dependent increase in sCD163 and IL-10 at 48 h with AVR-48 treatment without significant change in TNF-α level. However, LPS-treated cells showed an increase in sCD163 and IL-10 along with increased concentration of TNF-α at the 6-h to 48-h timepoints. This further confirms that increased IL-10 concentration by AVR-48 is not exclusively via TLR4 activation, but the activation of CD163 contributed to this outcome as well. The decrease in sCD163 and TNF-α at 72 h after LPS+AVR-48 treatment demonstrated the consorted effort of CD163and TLR4-mediated IL-10 production to negatively regulate TNF-α and IL-6, bringing all cytokine levels close to control creating a feedback regulation. The increase in IL-10 and decrease in inflammatory cytokine IL-17A in mouse lung and blood corroborated with the in vitro results.
LPS is a known TLR4 agonist and a secreted toxin of the gram-negative bacteria, including P. aeruginosa that activates TLR4 via the MyD88 pathway, activating the nuclear transcription factor NFkB and the release of inflammatory cytokines TNF-α, IL-1β, IL-6 and other chemokines. These molecules trigger other immune cells to act together as the first line of defense to kill invading bacteria and other microbial pathogens via increased phagocytosis and enhanced intracellular killing. So, we believe that activation of both TLR4 and CD163 by AVR-48 to differentiate the macrophages to an intermediary phenotype might be the cause of the key results: increased phagocytosis and decreased bacterial load in the mouse model of P. aeruginosa infection as well in the in vitro THP-1 phagocytosis assay. AVR-48 by itself did not demonstrate any direct bactericidal activity, and the concentration where 90% of bacteria get killed (MIC 90 ) was ≥200 µg/mL when tested for several gram positive (MRSA, MSSA) and gram negative (P. aeruginosa, A. baumannii, E. coli, K. pneumonia) using a broth dilution assay (Supplementary Figure  S5). Surprisingly, when AVR-48 was combined with several of the SoC broad-spectrum antibiotics-meropenem, colistin, ciprofloxacin-the MIC 90 of the antibiotics were lowered more than four-to tenfold, enhancing the anti-microbial activity and supporting the concept of synergy/additive activity [13] between these two modalities with an unknown mechanism that needs further investigations.
In summary, using primary spleen derived mononuclear cells, we showed that a small molecule AVR-48 derived from chitin with N-acetyl glucosamine as a core structure has a unique immunomodulating activity that restores the balance between activation and inhibition of innate immune response when exposed to LPS or to gram-negative bacteria. AVR-48 binds selectively to both TLR4 and CD163 receptors present in monocytes/macrophages with sub-micromolar IC 50 and has no binding affinity for T cells or B cells. The net effect of the binding of AVR-48 to both TLR4 and CD163 receptors was an increase in percentage of intermediate macrophages population (M int ) positive for both HLA-DR antigen and mannose receptor CD206, rendering both phagocytic and anti-inflammatory activities when tested in both in vitro and in vivo scenario using gram-negative bacteria Pseudomonas aeruginosa. In an in vivo model of a P. aeruginosa-induced lung infection in mice, AVR-48 treatment produced a significant decrease in lung bacteria counts; inflammatory cytokine IL-17A showed improvement of gross lung pathology. Increased concentrations of the anti-inflammatory cytokine IL-10 was observed in both mouse lung and plasma. Moreover, we observed a synergy and additive effect of AVR-48 with several SoC antibiotics providing an opportunity for both mono and combination therapy a during bacterial lung infection.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/immuno2040040/s1, Figure S1: Binding study of biotin conjugated AVR-48 to mouse monocytes; Figure S2: Binding of biotinylated conjugated AVR-48 to splenic monocytes, T cells and B cells; Figure S3: AVR-48 treatment increased resident monocytes in mouse spleen cells after 72 h; Figure S4: Flow cytometry data after treatment of AVR-48 to hPBMCs for 48 h; Figure