Abstract Introduction Infants born to mothers with a family history of allergy often display transiently low cord blood T cell (CBTC) protein kinase C ζ (PKCζ) levels, which are associated with an increased allergy risk. Our previous studies have shown that PKCζ is a key regulator of neonatal T cell development, promoting a Th1 cytokine bias. Maternal supplementation with ω-3 polyunsaturated fatty acids (PUFAs) during pregnancy enhanced neonatal PKCζ expression and protected against allergic sensitisation. The purpose of our investigation was to discover whether PKCζ is both a biomarker of allergy risk and a target for allergy prevention. Methods CBTC and a surrogate CBTC culture maturation model was used to examine the levels of PKCζ and cytokines produced, employing monoclonal antibodies and flow cytometry. The modulatory effects of ω-3 PUFAs was assessed by adding the fatty acids directly to the CBTC. Similarly, when examining the effects of PUFA metabolic products. The role of the fatty acid metabolism was examined by using inhibitors of the lipoxygenase and cyclooxygenase pathways. Results The data demonstrate that ω-3 PUFA treatment caused an increase in PKCζ expressed by the CBTC. Interestingly we found that while some PUFAs caused an increase, others not only failed to increase the PKCζ levels but induced a decrease in this expression. The increase in PKCζ in CBTC induced by ω-3 DHA was reflected in a transition of cells maturing away from a Th2 allergy-promoting cytokine propensity (i.e. Th1 cytokine profile predominance). Its metabolism through the lipoxygenase pathway was import for this effect and it was related to the generation of resolving-D1. Conclusion The findings identify PKCζ as a target in newborn T cell to prevent development towards a Th2-cytokine propensity and potentially allergic sensitisation. These expand the ‘hygiene hypothesis’ to a nutrient environment that regulates PKCζ expression. Funding Source Adelaide Scholarships International (ASI, 2016-2020), University of Adelaide; Invest for Success grant, Robinson Research Institute, The University of Adelaide. Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

The complement receptor immunoglobulin (CRIg), a key microbial pathogen phagocytosis-promoting receptor, responsible for intravascular clearance of bacteria, is purported to be expressed selectively on tissue-fixed macrophages such as Kupffer cells. However, recently it has been reported that neutrophils can also express functional CRIg following activation by inflammatory mediators. Monocytes have been reported not to express CRIg under non-activated conditions. Thus, investigations were undertaken to examine whether blood monocytes express CRIg under cell activation conditions and its role in anti-microbial immunity. Monocytes CRIg expression in whole human and mouse blood or peripheral blood mononuclear cells and purified monocytes using density gradient centrifugation or an affinity purification kit was examined using PE/FITC-labelled anti-CRIg monoclonal antibody and flow cytometry. Characterization of CRIg isoforms in monocytes was determined by the detection of CRIg mRNA transcripts and protein using RT-PCR and Western blot, respectively. Gene-edited CRIg– and CD18– monocytic THP-1 cell lines were generated to assess the role of CRIg and CD18 in cell adhesion, phagocytosis, and microbial killing. Functional assays were performed using Staphylococcus aureus as a model pathogen. CRIg was constitutively expressed, dynamically, on the surface of human and mouse blood monocytes. All three human monocyte subpopulations expressed CRIg, equally. The inability to demonstrate expression on monocytes cell surface by previous studies can be explained by its lability during blood storage and loss during monocyte isolation steps. Interestingly of the monocyte subpopulations only the classical and intermediate but not the non-classical showed a loss of CRIg expression. The data showed that loss from the surface was most likely due to relocation of the receptor intracellularly. Monocytes expressed 6 different CRIg mRNA transcripts and immunoreactive isoforms. Using CRIg– and CD18– THP-1 monocytic cells, we found that both CRIg and CD18 (CR3/CR4) were critical for cell adhesion, but for phagocytosis and killing of S. aureus, either receptor was independently effective. The data provide compelling evidence that monocytes express functional CRIg, relevant to the cells’ anti-microbial role of the ‘wandering’ phagocyte and consolidate a view that CRIg is widely expressed in our phagocytic cell system, similar to the classical complement receptors CR3 and CR4.

Neutrophils are the first line of defense against fungal infection and other microbial pathogens. Through alternative pathway of complement activation in innate immunity, complement fragments (opsonin) generated promote phagocytosis of the yeast forms of fungal pathogens such as Candida albicans. However, when complement levels are limited, through consumption in disease and inflammation or where microbial pathogens employ mechanisms to degrade or inhibit complement components, phagocytosis may still proceed via innate receptors, in the absence of effective opsonisation. Mechanisms of innate phagocytosis of fungi via neutrophils remain ill-defined and controversial. We have addressed this issue and demonstrated that neutrophils show very little phagocytosis of zymosan. Interestingly, activation or priming of neutrophils with phorbol-12-myristate-13-acetate, N-formyl-methionyl-leucyl-phenylalanine, arachidonic acid, tumor necrosis factor, and granulocyte macrophage-colony stimulating factor led to significant phagocytosis of the fungi. The result also demonstrated that priming led to a significant increase in the expression of the neutrophil Dectin-1, a receptor for β-glucan on zymosan. The increased phagocytosis induced by priming could be blocked with an anti-Dectin-1 antibody as well as the soluble β-glucan, laminarin, suggesting the importance of Dectin-1 in the phagocytosis of zymosan. The priming mechanism for phagocytosis and upregulation of Dectin-1 was examined using pharmacological inhibitors of protein kinase C (PKC) and p38 MAP kinase activation. The results showed that priming occurs in a PKC- p38-dependent as well as a PKC-independent p38-dependent manner. The findings demonstrate a highly regulated innate immune mechanism that potentially rapidly tackles microbial pathogens invading the bloodstream and tissues.

Autosomal-recessive pathogenic variants in Actin-related protein 2/3 complex subunit 1B (ARPC1B) result in an inborn error of immunity associated with eczema, thrombocytopenia, leukocytoclastic vasculitis and colitis. ARPC1B deficiency leads to impaired actin filament branching, affecting cytoskeletal processes in leukocytes, including migration, adhesion, endocytosis, and phagocytosis. This report presents two index cases from different families, who despite sharing an identical germline homozygous ARPC1B splice-site variant, ARPC1Bc.64+2T> A, had distinct clinical phenotypes including age of onset of symptoms and clinical manifestations. In addition, differences were observed in their immunological profiles including neutrophil chemotaxis, upregulation of complement receptor expression as well as B cell maturation and responses to EBV transformation. Further molecular investigation suggests the downstream cryptic splice-site activated by the mutation permits ‘leaky splicing’, enabling trace expression of wildtype ARPC1B. This supports a less severe deficiency in ARPC1B activity, rendering a less severe phenotype in one case compared with the other due to differential transcriptional activity of wildtype ARPC1B.

Liver fibrosis represents the liver’s adaptive response to sustained and intense inflammation triggered by diverse insults, ranging from dietary factors to infectious agents. Although early-stage fibrosis can be reversible, unchecked progression often leads to cirrhosis and hepatocellular carcinoma. Therapeutic options remain limited, highlighting the pressing need for novel anti‑fibrotic agents. In this study, we evaluated the in vitro anti‑fibrotic effects of 7‑octenoic acid (7‑OCT), a bioactive constituent from Moringa oleifera Lam. Human hepatic stellate LX‑2 cells were stimulated with TGF‑β1 (10 ng/mL) in the presence or absence of 7‑OCT. Fibrotic marker expression was quantified at both the mRNA and protein levels. Comprehensive proteomic profiling and subsequent in silico docking analyses were performed to identify putative molecular targets and clarify mechanisms of action. Our results demonstrate that 7‑OCT significantly suppresses the expression of α‑smooth muscle actin (α‑SMA), collagen type I, and matrix metalloproteinase‑9 (MMP‑9) by downregulating transforming growth factor beta receptor 1 (TGFBR1) expression and inhibiting mothers against decapentaplegic homolog 3 (SMAD3) phosphorylation. Proteomic data implicates focal adhesion pathway proteins in mediating these effects. Collectively, these findings suggest that 7‑OCT is a promising lead compound for the development of anti‑fibrotic therapies.

A significant number of babies born to women with a family history of allergy have transiently low levels of cord blood T cell protein kinase C (PKC)ζ, and increased incidence of allergic sensitisation by 2.5 y of age. We have provided compelling evidence for the role of PKCζ in regulating neonatal T cell development toward a Th1 cytokine propensity. We have now defined the transient deficient period after birth and show that interaction with nutrients during this period can increase and ‘normalise’ the PKCζ levels in immature T cells. In particular, we have demonstrated that polyunsaturated fatty acids are effective modulators of PKCζ levels. The results also identify which fatty acid structures are critical for achieving these changes and the fatty acid metabolic products responsible for their effects. These polyunsaturated fatty acids effects were reflected in the development of neonatal T cells from an immature allergy Th2 cytokine profile to a mature Th1 cytokine bias. The data identify a protein amenable to modulation by nutrients early after birth, representing a modification of the ‘hygiene hypothesis’ for preventing allergic sensitisation. Women’s and Children’s Research Foundation: Perveen_2025 Immune Response Regulation: Molecular Mechanisms (IRM)

While Moringa oleifera Lam. (MO) extracts are known to have various bioactive properties, including anti-inflammatory properties, the components responsible still remain to be identified. This study explores the protective effects of the MO component, 7-octenoic acid (7OCT) in LPS-stimulated THP-1 macrophage inflammatory responses. The compound significantly downregulated the production of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, as well as the expression of inflammation-related genes NFKB1, PTGS2, and NOS2. Additionally, it inhibited the nuclear translocation of NF-κB p65, a key transcription factor of inflammatory signaling cascade. Effects on oxidative stress showed that 7OCT inhibited LPS-induced NADPH oxidase 2 (NOX2) component genes including CYBB, CYBA, NCF1, NCF2, and NFE2L2, along with phosphorylated NOX2 and p47phox proteins. The compound reduced the expression of TP53, BAX, CASP3, and CASP7, while enhancing BCL2 expression and Bcl-2 protein levels, suggesting an effect on apoptosis. Decreased levels of BAX, caspase-3, and cleaved caspase-3 proteins further confirmed its anti-apoptotic effect. Our findings suggest that 7OCT exhibits strong anti-inflammatory, antioxidant, and anti-apoptotic properties.

Liver fibrosis, characterized by excessive extracellular matrix deposition, is driven by activated hepatic stellate cells (HSCs). Due to the limited availability of anti-fibrotic drugs, the research on therapeutic agents continues. Here we have investigated Moringa oleifera Lam. (MO), known for its various bioactive properties, for anti-fibrotic effects. This study has focused on 1-phenyl-2-pentanol (1-PHE), a compound derived from MO leaves, and its effects on LX-2 human hepatic stellate cell activation. TGF-β1-stimulated LX-2 cells were treated with MO extract or 1-PHE, and the changes in liver fibrosis markers were assessed at both gene and protein levels. Proteomic analysis and molecular docking were employed to identify potential protein targets and signaling pathways affected by 1-PHE. Treatment with 1-PHE downregulated fibrosis markers, including collagen type I alpha 1 chain (COL1A1), collagen type IV alpha 1 chain (COL4A1), mothers against decapentaplegic homologs 2 and 3 (SMAD2/3), and matrix metalloproteinase-2 (MMP2), and reduced the secretion of matrix metalloproteinase-9 (MMP-9). Proteomic analysis data showed that 1-PHE modulates the Wnt/β-catenin pathway, providing a possible mechanism for its effects. Our results suggest that 1-PHE inhibits the TGF-β1 and Wnt/β-catenin signaling pathways and HSC activation, indicating its potential as an anti-liver-fibrosis agent.

Reduced/deficient expression of Protein Kinase C (PKC)ζ in Cord blood (CB) T cells is associated with allergy development in children and a propensity to maintain an immature T-helper (Th)2 cytokine profile. In addition, other PKC isozymes are also low in CBTCs. Since previous studies have reported that cord blood/neonatal monocyte and neutrophil functions are significantly lower than cells from adults, it was of interest to see if the CBTC PKC levels were reflected in CB monocytes and neutrophils. Compared to adult blood, CB expresses low levels of PKCα, β2, ε, θ, μ, ζ and λ/ι in monocytes and PKCα, β2, η, θ, μ, ζ and λ/ι in neutrophils. The T-cell PKCζ levels were positively correlated with levels in CB monocytes but not in neutrophils. However, neither the monocytes nor the neutrophil PKCζ were associated with T-cell development towards a Th1 or Th2 cytokine propensity, based on the production of interferon-gamma and interleukin-4 in response to phytohemagglutinin and phorbol myristate acetate. The results demonstrate that some newborn babies display a deficiency in PKC isozymes in monocytes and neutrophils, as reported for T cells. However, unlike T cells, the PKCζ levels of the phagocytes did not correlate with regulation of development towards a Th1 or Th2 cytokine phenotype.

Liver fibrosis, a consequence of chronic liver damage or inflammation, is characterized by the excessive buildup of extracellular matrix components. This progressive condition significantly raises the risk of severe liver diseases like cirrhosis and hepatocellular carcinoma. The lack of approved therapeutics underscores the urgent need for novel anti-fibrotic drugs. Hepatic stellate cells (HSCs), key players in fibrogenesis, are promising targets for drug discovery. This study investigated the anti-fibrotic potential of Citrus hystrix DC. (KL) and its bioactive compound, β-citronellol (β-CIT), in a human HSC cell line (LX-2). Cells exposed to TGF-β1 to induce fibrogenesis were co-treated with crude KL extract and β-CIT. Gene expression was analyzed by real-time qRT-PCR to assess fibrosis-associated genes (ACTA2, COL1A1, TIMP1, SMAD2). The release of matrix metalloproteinase 9 (MMP-9) was measured by ELISA. Proteomic analysis and molecular docking identified potential signaling proteins and modeled protein–ligand interactions. The results showed that both crude KL extract and β-CIT suppressed HSC activation genes and MMP-9 levels. The MAPK signaling pathway emerged as a potential target of β-CIT. This study demonstrates the ability of KL extract and β-CIT to inhibit HSC activation during TGF-β1-induced fibrogenesis, suggesting a promising role of β-CIT in anti-hepatic fibrosis therapies.

Despite statements to the effect that you cannot measure serum cytokines, there have been numerous studies attempting to use serum cytokine measurements as biomarkers of disease activity and severity, relating to infection, autoimmunity, acute and chronic inflammatory diseases, and cancer. The approach is thwarted by assay inaccuracies, sensitivity and standardisation. This extends to the lack of information on serum sample handling and storage. Further setbacks are encountered from the lack of ability to generate appropriate normal intervals and cut-offs relevant to disease prognosis.

Medical diagnostic laboratories have come under further scrutiny to ensure quality standards of their service and external quality assurance (EQA) programs involving multiple laboratories have been used to gauge this quality based on a consensus. However, because of the geographical distances within a country or internationally, cell surface marker expressions may change due to time delays and transport temperatures. Attention was given to this issue some decades ago and hence requires a reevaluation in consideration of updated methods, reagents and instruments for flow cytometry and phenotyping. We have undertaken an extensive study to examine the effects of various conditions on blood storage akin to that experienced by patient samples as well as EQA programs, examining expression of lymphocyte surface markers, CD3, CD4, CD8, CD2, CD19, CD20, CD16/56 and HLADR. Assessment of lithium-heparin anticoagulated whole blood showed an increase in percentage of CD3 + and CD8 + T cells and a decrease in CD16/56 + NK cells after storage at room temperature (RT) for 24 and/or 48 h. In comparison, storage at 4 degrees C led to a decrease in percentage of CD4 + and increase in percentage of CD8 + cells. The low temperature also caused an increase in percentage of B cells (CD19 + , CD20 + ). While storage at RT did not alter levels of HLA-DR + CD3 + T cells, there was a significant increase in percentage of these cells after 48 h. Changes were also seen at both temperatures when EDTA was used as an anti-coagulant. Assessment of blood treated with a stabiliser, normally used in the EQA samples (Streck Cell Preservative), reduced the range of lymphocyte subsets affected, with only CD2 + and CD20 + cells being significantly different at both temperatures, We conclude that 24-48 h storage/transport can affect the percentage of CD3 + , CD4 + T cells, CD8 + T cells, B cells, NK cells and HLADR + T cells which can be minimised by using the blood stabiliser as per EQA programs and we emphasise the need to adopt this in the processing of patients' blood samples.

A significant number of babies present transiently with low protein kinase C zeta (PKCζ) levels in cord blood T cells (CBTC), associated with reduced ability to transition from a neonatal Th2 to a mature Th1 cytokine bias, leading to a higher risk of developing allergic sensitisation, compared to neonates whose T cells have ‘normal’ PKCζ levels. However, the importance of PKCζ signalling in regulating their differentiation from a Th2 to a Th1 cytokine phenotype propensity remains undefined. To define the role of PKCζ signalling in the regulation of CBTC differentiation from a Th2 to a Th1cytokine phenotype we have developed a neonatal T cell maturation model which enables the cells to develop to CD45RA − /CD45RO + T cells while maintaining the Th2 immature cytokine bias, despite having normal levels of PKCζ. The immature cells were treated with phytohaemagglutinin, but in addition with phorbol 12‐myristate 13‐acetate (PMA), an agonist which does not activate PKCζ. This was compared to development in CBTC in which the cells were transfected to express constitutively active PKCζ. The lack of PKCζ activation by PMA was monitored by western blot for phospho‐PKCζ and translocation from cell cytosol to the membrane by confocal microscopy. The findings demonstrate that PMA fails to activate PKCζ in CBTC. The data show that CBTC matured under the influence of the PKC stimulator, PMA, maintain a Th2 cytokine bias, characterised by robust IL‐4 and minimal interferon gamma production (IFN‐γ), and lack of expression of transcriptional factor, T‐bet. This was also reflected in the production of a range of other Th2/Th1 cytokines. Interestingly, introduction of a constitutively active PKCζ mutant into CBTC promoted development towards a Th1 profile with high IFN‐γ production. The findings demonstrate that PKCζ signalling is essential for the immature neonatal T cells to transition from a Th2 to a Th1 cytokine production bias.

Actin-related protein 2/3 complex subunit 1B (ARPC1B) deficiency is an autosomal-recessive inborn error of immunity resulting in impaired actin cytoskeletal function. This condition presents with similar features to Wiskott-Aldrich Syndrome, including leukocytoclastic vasculitis, thrombocytopenia, eczema and colitis. An absence of ARPC1B protein leads to impaired actin filament formation, which is critical for leukocyte activity in mediating cytoskeletal processes, including migration, adhesion, endocytosis, and phagocytosis. We present two index cases from different families, who despite sharing an identical homozygous ARPC1B splice-site variant, had distinct clinical phenotypes including age of onset of symptoms and clinical manifestations. In addition, differences were observed in their immunological profiles including neutrophil chemotaxis, upregulation of complement receptor expression as well as the B cell responses to EBV transformation. We postulate that the observed phenotypic differences are likely due to reversion mosaicism, which restores or maintains a level of residual function in particular immune compartments.

Background: C-reactive protein (CRP) is commonly performed, whereas cytokine testing is limited to research. Aims: To determine CRP correlation to cytokines IL-6, IL-1 beta and TNF-alpha. Results: Consecutive samples (n = 307) were collected over 24 h. Ninety-six patients (31%) had acute infections, and 23 patients (7.5%) had autoimmune or inflammatory disease presentations. A strong correlation between CRP and two IL-6 assays (r = 0.74 and r = 0.71; p < 0.001) was present. CRP did not correlate with IL-1 beta and TNF-alpha across the data set. Bacterial infection had a significantly higher CRP and IL-6 (p < 0.001), while only CRP was elevated in inflammatory and autoimmune diseases (p < 0.001). Discussion: CRP may be used as a surrogate marker of IL-6 levels in the routine diagnostic laboratories.

Background Infiltration of monocyte-derived macrophages into the synovial tissue (ST) is a hallmark of rheumatoid arthritis (RA) pathology. These macrophages promote inflammation, local joint effusion, and joint damage via the release of cytokines, oxygen reactive species, and tissue damaging enzymes. However, balancing these, are the ‘regulatory’ macrophages with inflammation-resolving properties, characterised by expression of CD206 and MerTK, dominant within the ST of healthy individuals as well as RA patients in remission (1). Indeed, these cells are believed to actively contribute to the maintenance of remission. Macrophages are known to exhibit remarkable phenotypic plasticity and understanding the role of this characteristic in regulating inflammation and pathology remains a major challenge, as does the characterization of factors in the microenvironment such as the synovium that control such macrophage characteristics. Importantly, whether the infiltrating, inflammatory macrophages of the RA ST similarly exhibit such phenotypic plasticity, and whether this occurs during the process of reaching remission, remains to be studied. Objectives We investigated the phenotypic plasticity of inflammatory synovial macrophages from patients with RA in vitro, investigating their ability to convert from an inflammatory macrophage population into ‘regulatory’ CD206 + MerTK + macrophages. These findings will provide a proof-of-concept as to the utility of these macrophage for a cell-based therapy in resolving inflammation in patients with RA, and will likely extend our understanding of the mechanisms of action of currently used therapeutics. Methods Synovial fluid (SF) mononuclear cells were obtained from patients with active early RA (<1 year; fulfilling 2010 ACR/EULAR classification criteria). Cryopreserved SFMCs were cultured for 48hr in the presence of 10 ng/mL interferon(IFN)γ, 50 ng/mL dexamethasone, 10 μg/mL Infliximab, or diluent. Following culture, cells were immunostained and analysed using a Beckman Coulter CytoFLEX flow cytometer and FlowJo software. SF macrophages were characterised by expression of CD14, CD45, CD68 (Figure 1A), and proportions of CD206 + MerTK + macrophages measured. Figure 1. Synovial fluid CD68 + macrophage plasticity in vitro. (A) Gating strategy depicting CD68 + CD45 + CD14 + SF macrophage determination. (B) Proportions of CD206 and MerTK-expressing SF macrophages after 48hr culture in the presence of 10 ng/mL IFNγ, 50 ng/mL dexamethasone or 10 µg/mL Infliximab, or absence. Data are representative of 5 individual experiments. Data were analysed by two-way ANOVA followed by Dunnett’s multiple comparison test, *p<0.05. Results Prior to culture, the CD68 + macrophage populations present in SF were found to be predominantly CD206 - MerTK - . After 48 hours of culture, in the absence of any stimulus, there was an increase in proportions of CD206 + MerTK + macrophages. Treatment with either dexamethasone or anti-TNF (Infliximab) resulted in a further increase in proportions of CD206 + MerTK + , M2-like macrophages. In contrast, culture with IFNγ induced a reduction in this population. Importantly, we found that the generated CD206 + MerTK + macrophages were phenotypically stable in culture following removal of these differentiating agents. Conclusion Our findings demonstrate that inflammatory SF cells are indeed able to polarise to regulatory, CD206 + MerTK + macrophages in vitro . The findings provide further mechanistic insights into the basis for the therapeutic benefits of glucocorticoids and TNF inhibitors, as well as providing initial proof-of-concept in the use of regulatory macrophages as a cellular-based therapy or therapeutic target for patients with RA. References [1]Alivernini S, MacDonald L, Elmesmari A, et al., Distinct synovial tissue macrophage subsets regulate inflammation and remission in rheumatoid arthritis. Nature Medicine. 2020;26(8):1295-306 10.1038/s41591-020-0939-8. Disclosure of Interests None declared

Autosomal-dominant cryopyrin-associated periodic syndromes (CAPS) are a group of autoimmune inflammatory conditions characterised by the constitutive activation of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome and excessive interleukin (IL)-1β production. This is typically associated with heterozygous pathogenic variants in the NLRP3 gene. However, owing to a lack of information in the literature or variant databases, not all missense variants are classified pathogenic or likely pathogenic when applying the American College of Medical Genetics and Genomics (ACMG) guidelines established for genomic reporting, particularly if extremely rare or novel. We present the case of a family with multiple members with a CAPS phenotype, carrying a rare and poorly described NLRP3 missense substitution. To verify pathogenicity, we sought to establish a novel diagnostic NLRP3 inflammasome assay workflow to examine gain-of-function activity of primary peripheral blood mononuclear cells from family members with or without the variant, in comparison with healthy donor controls. This was achieved through activating the inflammasome with lipopolysaccharide and adenosine triphosphate, applying a rapid bioluminescent capsase-1 activity assay, measuring IL-1β production, with normalisation to CD14+ monocyte content. We demonstrated a perfect binary functional interpretation, as all specimens positive for the NLRP3 variant showed a hyperactive NLRP3 inflammasome response.