All procedures were approved by the West Virginia University Institutional Animal Care and Use Committee (Protocol #:1708008935) and conducted in accordance with the recommendations from the Guide for the Care and Use of Laboratory Animals by the National Research Council (NRC, 2011).

Breeding pairs of C57BL/6 (WT) or IL-27Rα^-/- (KO) mice on a C57BL/6 genetic background were purchased from Jackson Laboratories (Bar Harbor, ME) and maintained under specific pathogen-free conditions in the vivarium at West Virginia University Health Sciences Center. Mice were maintained on a 12-h light/dark cycle and were fed/watered ad libitum. Male and female pups (~300) were used for experimental infection. Blood and tissues were collected from mice at the appropriate age by humane procedures. Euthanasia procedures involved isoflurane inhalation at 4-5% induction.

Escherichia coli isolates with serotype O1 are invasive and frequently express the K1 capsule, a virulence factor associated to neonatal meningitis, bacteremia, and septicemia (29). We established a murine neonatal sepsis model using Escherichia coli strain O1:K1:H7 (ATCC 11775) obtained from the ATCC (Manassis, VA, USA). The bacteria were grown in Luria broth from a single colony isolated on Tryptic Soy agar (TSA). To prepare infectious inoculums, the bacteria were enumerated as described previously (21). Neonatal pups (n=3 per group [control vs. infected] per genotype [WT vs. KO]) at 4 days were inoculated subcutaneously in the scapular region with E. coli O1:K1:H7 using a 28-gauge insulin needle as described previously (20, 21). The bacteria were washed with PBS, centrifuged at 2,000 x g for 5 min, and suspended in a volume of PBS equivalent to an inoculum of 50 μL/mouse. The target inoculum was 2×10⁵ CFUs per mouse and actual inoculums as determined by standard plate counts, ranged from 2-3x10⁵ CFUs per mouse. Vehicle (PBS)-received pups were identified from E. coli-infected pups using a tail snip. The weights of mice were recorded immediately prior to infection, and at 24 h immediately prior to euthanasia. Blood glucose levels were measured using an AlphaTrack3 blood glucose monitoring system (Zoetis, MI, USA). All downstream experiments were associated with the same pre-identified mice. Spleens isolated from pups were placed in PBS on ice. For some experiments, spleens were placed in 4% paraformaldehyde for further histopathological studies. Blood was deposited in tubes that contained 5 μL of 500 mM ethylenediamine tetraacetate acid (EDTA) and placed on ice. Serum was collected by centrifuging at 2,000 x g for 10 min. The bacterial burden in the spleen was enumerated by serial dilution and standard plating on TSA.

Spleens were homogenized in TRI Reagent^® (Molecular Research Center, Cincinnati, OH). According to the commercial product protocol, the upper aqueous layer following phase separation was mixed with an equal volume of 75% ethanol and transferred to E.Z.N.A.^® RNA isolation columns (Omega Biotek, GA, USA). The manufacturer’s instructions were followed to complete tissue RNA isolation using E.Z.N.A.^® RNA isolation kit (Omega Biotek, GA, USA). RNA from cells or tissue was quantified using a nanodrop. First-strand cDNA was synthesized using the iScript™ cDNA synthesis kit (Bio-Rad, CA, USA). Quantitative PCR reactions included cDNA diluted four-fold, gene-specific TaqMan^® primer probe sets (ThermoFisher, MA, USA), and iQ™ Supermix (Bio-Rad, CA, USA). Cycling was performed in triplicate using a StepOnePlus™ Real-Time detection system (ThermoFisher, MA, USA). Gene-specific amplification as an internal reference gene and expressed as log₂ relative gene expression compared to control spleen using the formula 2^-ΔΔCt. CXCR2 (ID: Mm. PT. 58. 12102629) and CXCL2 (ID: Mm. PT. 58.10456839) assays were purchased from Integrated DNA Technologies (Coralville, IA, USA). The β-actin assay (ID: mM01205647_g1) was purchased from ThermoFisher (MA, USA).

Splenocytes (5x10⁵) from infected WT and KO animals isolated as described above were added onto the membrane of the 24-transwell insert (3 μm pore size) in a volume of 100 µL DMEM. A volume of 600 µL complete DMEM with or without 0.025 ng/mL CXCL2 chemoattractant was carefully added to the well. The transwell culture was incubated at 37°C with 5% CO₂. Images of migrated cells in the medium of the bottom chamber were taken at 1 and 4 h post-incubation using an Evos imaging system (Thermo Fisher Scientific, Massachusetts, USA). Average of 4 picture fields at 10x magnification were taken for each condition and cell counts were determined using ImageJ automated software (30).

Four-day old WT (n=5) and KO (n=5) pups infected for 24 h were humanely euthanized and the spleens collected. To obtain sufficient cells for downstream analysis, spleens were pooled based on infection status (control vs. infected). Single-cell suspensions were generated by dissociating the spleens through a 40 µm strainer and centrifugation at 350 × g for 5 min. Cells were resuspended in 0.5 mL of ammonium/chloride/potassium erythrocytes lysis buffer (ThermoFisher Scientific, Massachusetts, USA) and incubated on ice for 5 min, followed by centrifugation at 350 × g for 5 min. Mononuclear leukocytes were enriched from splenocytes using Optiprep™ (Sigma-Aldrich, Illinois, USA) density gradient centrifugation as described previously and scaled down four-fold to accommodate the smaller number of cells in neonatal spleens (21, 31). Neutrophils were isolated from splenocytes with magnetically labeled anti-Ly6B.2 (7/4) microbeads and immunomagnetic selection using Miltenyi Biotec isolation reagents (Miltenyi Biotec, Bergisch Gladbach, Germany) as described previously (20, 32) Splenocytes, monocytes and neutrophils were counted using automated cell countess 3 (Invitrogen, Massachusetts, USA) used as described in downstream approaches. The enrichment of mononuclear cells and neutrophils was assessed by immunolabeling for CD11b, Ly6C, and Ly6G, respectively.

Mononuclear cells from WT and KO pups of seven day old were isolated as described above and 1×10⁶ cells seeded per well in a 6 well plate. These cells were treated with CXCL2 0.1µg/mL or 1µg/mL for 8 h. Cells were harvested, and RNA was extracted to check the gene expression of CXCR2.

5×10⁵ cells splenocytes or mononuclear cells were mixed with Fc receptor blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) and held at room temperature for 10 min prior to cell surface marker labeling with fluorochrome-conjugated antibodies. In addition to Live/Dead stain (FVS780, BD Biosciences, CA, USA), antibodies used for the panel included CD11b (BV786) and Ly6C (PE) from BD Biosciences and CXCR2 (APC) from Miltenyi Biotec. All antibodies were diluted according to manufacturer specifications. Immunolabeling was performed for 45 min on ice protected from light. Cells were washed, resuspended in 4% paraformaldehyde (PFA; ThermoFisher Scientific, MA, USA), and stored at 4 °C until flow cytometric analysis. All samples were analyzed on a LSRFortessa instrument (BD Biosciences) using FCS Express version 7.0 software. A minimum of 30,000 events were analyzed per sample.

Spleens were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at a thickness of 3 μm, and then stained with hematoxylin & eosin (H&E) solution according to standard procedures performed by the Electron Microscopy Histopathology and Tissue Bank Core Facility at West Virginia University. Histopathologic changes were observed by light microscopy (Olympus slide scanner, Japan). Organ damage was evaluated by measuring aspartate aminotransferase (Cayman, MI, USA) in the serum according to manufacturer’s protocol.

All data sets were analyzed with the appropriate parametric or nonparametric test as indicated in the figure legend using Prism 8 (GraphPad, San Diego, CA). The threshold for statistical significance was set at alpha = 0.05.

To understand the impact of IL-27 on regulation of the neonatal host response to infection, we previously analyzed the transcriptome of the neonatal spleen during K1-encapsulated E. coli-induced sepsis in WT and KO mice (25). KO mice do not express a functional receptor and cannot respond to IL-27. Many genes for cytokines, chemokines, and their receptors were increased in WT animals during infection, consistent with pronounced inflammation, but did not increase significantly in KO pups ( Figure 1A ) (25). In contrast, CXCR2 expression was upregulated significantly in KO neonates during infection but failed to increase in WT neonates ( Figures 1A, B ) (25). To further confirm these findings in new sets of animals, WT and KO neonates were infected on day 4 with E. coli and the spleens collected at 24 h post-infection to determine CXCR2 gene expression. CXCR2 expression levels in the KO neonates were significantly higher during infection ( Figure 1C ). WT neonates downregulated CXCR2 expression levels in the spleen upon E. coli infection ( Figure 1C ).

Transcriptome data showed that WT neonates had very high expression levels of numerous chemokines during infection ( Figure 2A ) (25). Among them CXCL2 is one of the high- affinity binding ligands for CXCR2 receptor (33). Gene expression data from the spleens of WT and KO neonates confirmed that during infection, CXCL2 chemokine levels were highly elevated in the WT ( Figures 2B, C ) whereas KO had moderately elevated expression ( Figure 2C ). In contrast to the higher levels of chemokine CXCL2 levels, the receptor expression is very low in the WT infected pups ( Figures 1 , 2 ). In the spleen, although IL-27 receptor KO infected pups had moderate expression levels of chemokine CXCL2, its receptor CXCR2 levels were high ( Figures 1 , 2 ).

Chemotaxis of myeloid cells from the blood and bone marrow is an important process to coordinate immune responses within the spleen, enabling host response to infection and maintenance of tissue homeostasis (34). This chemotaxis is regulated by various factors, including chemokine receptors on cells and chemokines expressed by cells at the target tissues (35). To determine if increased CXCR2 expression on KO splenocytes promoted enhanced migration, we measured chemotactic migration toward CXCL2 using an in vitro Boyden chamber assay ( Figure 3A ). WT and KO neonatal pups were infected with E. coli and spleens were harvested at 24 h post-infection ( Figure 3A ). Splenocytes were isolated, placed in the top of the transwell, and cells migrated toward CXCL2 in the bottom well were measured by microscopy. The results demonstrated that infected KO neonatal splenocytes exhibited greater migration efficiency compared to infected WT neonatal cells over 1-4 h ( Figures 3B, C ).

CXCR2 is expressed by mixed phagocytic myeloid cells such as neutrophils, monocytes, and macrophages and mediates migration of inflammatory cells (36, 37). The role of CXCR2 in neutrophil recruitment to inflammatory sites has been well established and shown to control the magnitude of the macrophage-dependent inflammatory response (38, 39). It is also known to regulate monocyte recruitment and function (40). After confirming elevated levels of CXCR2 and migration efficiency of KO splenocytes infected neonatal mice, we investigated the specific cell type influencing CXCR2 expression levels in the spleen. As such, we isolated neutrophils and mononuclear cells from the spleens of control and infected WT and KO neonatal mice. Anti-Ly6B2 magnetic beads were used to enrich neutrophils, and monocytes were isolated by density gradient centrifugation (41, 42). We observed an approximate two-fold increase in CXCR2 expression in splenic neutrophils from WT pups during infection relative to uninfected controls ( Figure 4A ). In contrast, there was no change in CXCR2 expression during infection in neutrophils from KO pups ( Figure 4A ). Surprisingly, there was no significant change in the CXCR2 expression observed in splenic mononuclear cells from WT pups during infection ( Figure 4B ). However, a dramatic 32-fold increase of CXCR2 expression levels observed in the mononuclear cells of infected KO neonates relative to uninfected controls ( Figure 4B ).

It is known that overproduction of proinflammatory mediators or persistent exposure to ligand during severe sepsis leads to desensitization of CXCR2 receptor (43). We hypothesized that highly elevated expression levels of CXCL2 chemokine in the WT might be suppressing the expression of chemokine receptor on mononuclear cells during infection. To evaluate this, mononuclear cells were isolated from naïve WT and KO neonatal mice and stimulated with a range of CXCL2 concentrations ex vivo. The results demonstrated that mononuclear cells from both WT and KO pups exhibited elevated expression levels of CXCR2 when stimulated with 0.1 μg/mL of chemokine CXCL2 ( Figures 5A, B ). In contrast, at higher concentration of CXCL2 downregulated the expression of CXCR2 on murine neonatal mononuclear cells ( Figures 5A, B ). This suggests that mononuclear cells from IL-27Ra KO mice are not inherently programmed to express CXCR2 at higher levels than WT, and instead, respond to the environment during infection to modulate their receptor expression level ( Figure 5C ).

Recruitment of monocytes is essential for effective control and clearance of bacterial infections. Monocytes originate from progenitors in the bone marrow and traffic via the bloodstream to peripheral tissues (44, 45). Monocytes are classified mainly into two subsets with different biological functions based on chemokine receptor expression and the presence of specific surface molecules (46). In mice, classical Ly6C^hi monocytes are often referred to as inflammatory monocytes which are rapidly recruited to sites of infection and differentiate further into pro-and anti-inflammatory macrophages which can clear bacteria and promote tissue remodeling or injury (47). To further understand the abundance and phenotype of the monocyte population in the spleen, we isolated mononuclear cells from both WT and KO neonatal mice in the presence and absence of infection at 24 h post-infection for analysis by flow cytometry ( Supplementary Figure 1 ). We consistently observed a higher percentage of Ly6C⁺ mononuclear cells that expressed CXCR2 in the infected KO neonates than the WT neonatal pups. ( Figure 6 ). These findings mirror the CXCR2 gene expression profiles and suggest enhanced monocyte recruitment to sites of infection.

The spleen is the largest lymphoid tissue and plays an important role in the immune defense to invasive infection, particularly for encapsulated bacteria, and is a key contributor to the exaggerated inflammatory response that occurs during sepsis (48). To explore the potential impact of increased mononuclear cell recruitment in the context of bacterial clearance and tissue injury, we examined spleens harvested at 24 h post-infection from each genotype and treatment condition for bacterial burdens and histopathology. We observed that spleens from infected IL-27Rα KO neonates have well-maintained tissue architecture with minimal damage, in contrast to spleens from the infected WT neonates that demonstrated necrotic regions with shrunken and fragmented cells consistent with increased bacterial burdens ( Figures 7A, B ). Serum ALT and AST levels are a reliable diagnostic tool for sepsis and septic shock that aid in predicting mortality (49). Serum aspartate transaminase (AST) is an enzyme found in the liver, muscles, and other tissues; levels are typically low in the blood and increase following release from damaged cells (50, 51). Elevated levels of AST in the serum correlate with tissue damage or apoptosis, inflammatory liver disease, septic shock, skeletal muscle injury and severe myocardial ischemia (52). A hallmark of sepsis is dysfunction and damage to multiple organs (53, 54), and as such, we measured serum AST levels as an indicator of tissue damage. We found a significant increase in serum AST levels during infection of WT neonates. However, AST levels in KO neonates remained comparable to uninfected control pups ( Figure 7C ). This suggests more limited tissue damage in the KO neonates during infection, in agreement with histological analysis of the spleen. As additional measures of morbidity, we also observed significant weight loss ( Figure 7D ) and hypoglycemia ( Figure 7E ), in the infected WT neonates. Conversely, infected KO neonates demonstrated improved maintenance of weight and blood glucose ( Figures 7D, E ).