Nuclear factor kappa B (NF-κB) is a critical transcription factor involved in regulating cell activation, inflammation, and survival. The linear ubiquitin chain assembly complex (LUBAC) which consists of HOIL1, HOIP, and SHARPIN, catalyzes the linear ubiquitination of target proteins-a post-translational modification that is essential for NF-κB activation. Human germline pathogenic variants that dysregulate linear ubiquitination and NF-κB signaling are associated with immunodeficiency and/or autoinflammation including dermatitis, recurrent fevers, systemic inflammation and enteropathy. We previously identified MALT1 paracaspase as a novel negative regulator of LUBAC by proteolytic cleavage of HOIL1. To directly investigate the impact of HOIL1 cleavage activity on the inflammatory response, we employed a stable transduction system to express and directly compare non-cleavable HOIL1 with wild-type HOIL1 in primary HOIL1-deficient patient skin fibroblasts. We discovered that non-cleavable HOIL1 resulted in enhanced NF-κB signaling in response to innate stimuli. Transcriptomics revealed enrichment of inflammation and proinflammatory cytokine-related pathways after stimulation. Multiplexed cytokine assays confirmed a 'hyperinflammatory' phenotype in these cells. This work highlights the physiological importance of MALT1-dependent cleavage and modulation of HOIL1 on NF-κB signaling and inflammation, provides a mechanism for the autoinflammation observed in MALT1-deficient patients, and will inform the development of therapeutics that target MALT1 paracaspase and LUBAC function in treating autoinflammatory skin diseases.

Early-life viral infections are responsible for pulmonary exacerbations that can contribute to disease progression in young children with cystic fibrosis (CF). The most common respiratory viruses detected in the CF airway are human rhinoviruses (RV), and augmented airway inflammation in CF has been attributed to dysregulated airway epithelial responses although evidence has been conflicting. Here, we exposed airway epithelial cells from children with and without CF to RV in vitro. Using RNA-Seq, we profiled the transcriptomic differences of CF and non-CF airway epithelial cells at baseline and in response to RV. There were only modest differences between CF and non-CF cells at baseline. In response to RV, there were 1,442 and 896 differentially expressed genes in CF and non-CF airway epithelial cells, respectively. The core antiviral responses in CF and non-CF airway epithelial cells were mediated through interferon signaling although type 1 and 3 interferon signaling, when measured, were reduced in CF airway epithelial cells following viral challenge consistent with previous reports. The transcriptional responses in CF airway epithelial cells were more complex than in non-CF airway epithelial cells with diverse over-represented biological pathways, such as cytokine signaling and metabolic and biosynthetic pathways. Network analysis highlighted that the differentially expressed genes of CF airway epithelial cells' transcriptional responses were highly interconnected and formed a more complex network than observed in non-CF airway epithelial cells. We corroborate observations in fully differentiated air-liquid interface (ALI) cultures, identifying genes involved in IL-1 signaling and mucin glycosylation that are only dysregulated in the CF airway epithelial response to RV infection. These data provide novel insights into the CF airway epithelial cells' responses to RV infection and highlight potential pathways that could be targeted to improve antiviral and anti-inflammatory responses in CF.

Intravenous Immunoglobulin (IVIg) is used to treat autoimmune or inflammatory diseases, but its mechanism of action is not completely understood. We asked whether IVIg can induce interleukin-10 (IL-10) and reduce pro-inflammatory cytokine production in human monocytes, and whether this response is reduced in monocytes from people with an Fcγ receptor IIA (FcγRIIA) gene variant, which is associated with increased risk of inflammatory diseases and poor response to antibody-based biological therapy. IVIg increased IL-10 production and reduced pro-inflammatory cytokine production in response to bacterial lipopolysaccharide (LPS), which required FcγRI and FcγRIIB and activation of MAPKs, extracellular signal-regulated kinase 1/2 (ERK1/2), and p38. IL-10 production was lower and pro-inflammatory cytokine production was higher in monocytes from people with the FcγRIIA risk variant and the risk variant prevented IL-10 production in response to (IVIg+LPS). Finally, we show that IVIg did not induce MAPK activation in monocytes from people with the risk variant. Our results demonstrate that IVIg can skew human monocytes to an anti-inflammatory, IL-10-producing activation state, which is compromised in monocytes from people with the FcγRIIA risk variant. This research has profound implications for the use of IVIg because 25% of the population is homozygous for the FcγRIIA risk variant and its efficacy may be reduced in those individuals. In addition, this research may be useful to develop new therapeutic strategies to replace IVIg by cross-linking FcγRIs and FcγRIIBs to promote anti-inflammatory macrophage activation, independent of the FcγRIIA genotype.

Colonization with Clostridioides difficile occurs in up to half of infants under the age of 3 months, is strongly influenced by feeding modality and is largely asymptomatic. In spite of this, C. difficile's presence has been associated with susceptibility to chronic disease later in childhood, perhaps by promoting or benefiting from changes in infant gut microbiome development, including colonization with pathogenic bacteria and disrupted production of microbial bioactive metabolites and proteins. In this study, the microbiomes of 1554 infants from the CHILD Cohort Study were described according to C. difficile colonization status and feeding mode at 3-4 months of age. C. difficile colonization was associated with a different gut microbiome profile in exclusively breastfed (EBF) vs. exclusively formula fed (EFF) infants. EBF infants colonized with C. difficile had an increased relative abundance of Firmicutes and Proteobacteria, decreased relative abundance of Bifidobacteriaceae, greater microbiota alpha-diversity, greater detectable fecal short chain fatty acids (SCFA), and lower detectable fecal secretory Immunoglobulin A (sIgA) than those not colonized. Similar but less pronounced differences were seen among partially breastfed infants (PBF) but EFF infants did not possess these differences in the gut microbiome according to colonization status. Thus, breastfed infants colonized with C. difficile appear to possess a gut microbiome that differs from non-colonized infants and resembles that of EFF infants, but the driving force and direction of this association remains unknown. Understanding these compositional differences as drivers of C. difficile colonization may be important to ensure future childhood health.