454 - Modulating Acute and Chronic Systemic Pro-Inflammatory Cytokine and Chemokine Responses in Neonatal Sepsis

Publication Number: 2442.454

Sana Shamshad, Stony Brook Children's Hospital, Medford, NY, United States; Bahman Khalvatifahlyani, Renaissance School of Medicine at Stony Brook University, Stonybrook, NY, United States; Joyce Lin, Stony Brook Children's Hospital, Port Washington, NY, United States; John D Haley, Stony Brook Medicine, Stony Brook, NY, United States; Robert Woroniecki, Stony Brook Children's Hospital, Stony Brook, NY, United States; Esther M. Speer, Stony Brook University, Stony Brook, NY, United States

Background: Sepsis in newborns (SN) leads to high mortality and multi-organ injury. The sepsis-associated pathogenesis involves excessive systemic inflammation, oxidative stress, and tissue damage, which cannot be mitigated by antibiotics alone. Pentoxifylline (PTX), a methylxanthine derivative with anti-inflammatory properties, has potential in modulating inflammatory responses during sepsis, and may thus help to mitigate organ injuries associated with SN.

Objective: To determine PTX potential role in attenuating the systemic inflammatory response, we measured the time course of plasma cytokines and chemokines following neonatal lipopolysaccharide (LPS) exposure with and without PTX administration.

Design/Methods: C57BL/6J neonatal mice (n = 3-5 per group) were randomly injected intraperitoneally with 2 µg/g body weight ultrapure LPS (from E. coli 0111:B4) or saline on days 2 and 3. PTX (60 µg/g/day, IP) or saline was administered starting with the first LPS dose and continued for 14 days. Blood plasma was collected on days 4, 14, and 4 weeks. Cytokines and chemokines were measured by proximity extension assay using Olink® Target 48 Mouse Cytokine Panel.

Results: Neonatal LPS exposure triggered a strong and prolonged systemic inflammatory response. On day 4, the LPS group showed marked increases in pro-inflammatory cytokines, including IL-6, TNF, and IL-17f, along with elevated chemokines such as Cxcl1, Csf3, and Ccl4, and increases in regulatory cytokines (IL-10 and IL-22). By day 14, several cytokines, including IL-16, IL-17f, IL-22, and IFN-γ, remained elevated, indicating a persistent inflammatory state. At 4 weeks, most cytokine concentrations had returned toward baseline. Treatment with PTX abolished the early cytokine surge on day 4, reducing cytokine and chemokine concentrations to at or below those of controls, and prevented sustained elevations observed at later time points. Cytokine levels in the LPS+PTX group remained comparable to controls throughout the study, demonstrating that early PTX intervention effectively blocked both the acute and prolonged inflammatory responses induced by neonatal LPS exposure.

Conclusion(s): Neonatal LPS exposure triggers both immediate and sustained cytokine and chemokine responses, which may contribute to peripheral organ inflammation and potential long-term organ injury, while PTX administration effectively mitigates these systemic inflammatory changes.

Figure 1: Acute serum cytokine and chemokine concentrations in postnatal day 4 newborn mice after LPS exposure and their modulation with PTX
Mean serum concentrations (pg/mL) of cytokines and chemokines in control (CTL, blue line), LPS-exposed (red line), and LPS+PTX-exposed (green line) neonatal mice on postnatal day 4.
Analytes shown include:
Cxcl1 (C-X-C motif chemokine ligand 1),
Il6 (Interleukin-6),
Fgf21 (Fibroblast growth factor 21),
Csf3 (Colony stimulating factor 3 / G-CSF),
Cxcl2 (C-X-C motif chemokine ligand 2),
Csf1 (Colony stimulating factor 1 / M-CSF),
Ifng (Interferon gamma),
Ccl5 (C-C motif chemokine ligand 5 / RANTES),
Pdcd1lg2 (Programmed cell death 1 ligand 2),
Il1b (Interleukin-1 beta),
Il17f (Interleukin-17F),
Tnf (Tumor necrosis factor alpha),
Il22 (Interleukin-22),
Il10 (Interleukin-10),
Ccl12 (C-C motif chemokine ligand 12),
Ccl4 (C-C motif chemokine ligand 4),
Cxcl12 (C-X-C motif chemokine ligand 12),
Ccl22 (C-C motif chemokine ligand 22),
Il5 (Interleukin-5),
Cxcl9 (C-X-C motif chemokine ligand 9),
Il16 (Interleukin-16).

Figure 2: Serum cytokine and chemokine concentrations in 14-days old mice after neonatal LPS exposure and their modulation with PTX
Mean serum concentrations (pg/mL) of cytokines and chemokines on postnatal day 14 in control (CTL, blue line), LPS-exposed (red line), and LPS+PTX-exposed (green line) mice.
(see Figure 1 for list of analytes)


Figure 3: Serum cytokine and chemokine concentrations in 4-weeks old mice after neonatal LPS exposure and their modulation with PTX
Mean serum concentrations (pg/mL) of cytokines and chemokines at age 4 weeks in control (CTL, blue line), LPS-exposed (red line), and LPS+PTX-exposed (green line) mice.
(see Figure 1 for list of analytes)