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Autoimmune diseases, such as psoriasis and arthritis, show a patchy distribution of inflammation despite systemic dysregulation of adaptive immunity. Thus, additional tissue-derived signals, such as danger-associated molecular patterns (DAMPs), are indispensable for manifestation of local inflammation. S100A8/S100A9 complexes are the most abundant DAMPs in many autoimmune diseases. However, regulatory mechanisms locally restricting DAMP activities are barely understood. We now unravel for the first time, to our knowledge, a mechanism of autoinhibition in mice and humans restricting S100-DAMP activity to local sites of inflammation. Combining protease degradation, pull-down assays, mass spectrometry, and targeted mutations, we identified specific peptide sequences within the second calcium-binding EF-hands triggering TLR4/MD2-dependent inflammation. These binding sites are free when S100A8/S100A9 heterodimers are released at sites of inflammation. Subsequently, S100A8/S100A9 activities are locally restricted by calcium-induced (S100A8/S100A9)2 tetramer formation hiding the TLR4/MD2-binding site within the tetramer interphase, thus preventing undesirable systemic effects. Loss of this autoinhibitory mechanism in vivo results in TNF-α-driven fatal inflammation, as shown by lack of tetramer formation in crossing S100A9-/- mice with 2 independent TNF-α-transgene mouse strains. Since S100A8/S100A9 is the most abundant DAMP in many inflammatory diseases, specifically blocking the TLR4-binding site of active S100 dimers may represent a promising approach for local suppression of inflammatory diseases, avoiding systemic side effects.

Calgranulin genes (S100A8, S100A9 and S100A12) play key immune response roles in inflammatory disorders, including cardiovascular disease. Long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) may have systemic and adipose tissue-specific anti-inflammatory and cardio-protective action. Interactions between calgranulins and the unsaturated fatty acid arachidonic acid (AA) have been reported, yet little is known about the relationship between calgranulins and the LC n-3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). We explored tissue-specific action of calgranulins in the setting of evoked endotoxemia and n-3 PUFA supplementation. Expression of calgranulins in adipose tissue in vivo was assessed by RNA sequencing (RNASeq) before and after n-3 PUFA supplementation and evoked endotoxemia in the fenofibrate and omega-3 fatty acid modulation of endotoxemia (FFAME) Study. Subjects received n-3 PUFA (n = 8; 3600mg/day EPA/DHA) or matched placebo (n = 6) for 6-8 weeks, before completing an endotoxin challenge (LPS 0.6 ng/kg). Calgranulin genes were up-regulated post-LPS, with greater increase in n-3 PUFA (S100A8 15-fold, p = 0.003; S100A9 7-fold, p = 0.003; S100A12 28-fold, p = 0.01) compared to placebo (S100A8 2-fold, p = 0.01; S100A9 1.4-fold, p = 0.4; S100A12 5-fold, p = 0.06). In an independent evoked endotoxemia study, calgranulin gene expression correlated with the systemic inflammatory response. Through in vivo and in vitro interrogation we highlight differential responses in adipocytes and mononuclear cells during inflammation, with n-3 PUFA leading to increased calgranulin expression in adipose, but decreased expression in circulating cells. In conclusion, we present a novel relationship between n-3 PUFA anti-inflammatory action in vivo and cell-specific modulation of calgranulin expression during innate immune activation.

The S100A8/S100A9 heterodimer calprotectin (CP) functions in the host response to pathogens through a mechanism termed “nutritional immunity.” CP binds Mn ²⁺ and Zn ²⁺ with high affinity and starves bacteria of these essential nutrients. Combining biophysical, structural, and microbiological analysis, we identified the molecular basis of Mn ²⁺ sequestration. The asymmetry of the CP heterodimer creates a single Mn ²⁺-binding site from six histidine residues, which distinguishes CP from all other Mn ²⁺-binding proteins. Analysis of CP mutants with altered metal-binding properties revealed that, despite both Mn ²⁺ and Zn ²⁺ being essential metals, maximal growth inhibition of multiple bacterial pathogens requires Mn ²⁺ sequestration. These data establish the importance of Mn ²⁺ sequestration in defense against infection, explain the broad-spectrum antimicrobial activity of CP relative to other S100 proteins, and clarify the impact of metal depletion on the innate immune response to infection.

Immune cells harboring somatic mutations reportedly infiltrate cancer tissues in patients with solid cancers and accompanying clonal hematopoiesis. Loss‐of‐function TET2 mutations are frequently observed in clonal hematopoiesis in solid cancers. Here, using a mouse lung cancer model, we evaluated the activity of Tet2‐deficient immune cells in tumor tissues. Myeloid‐specific Tet2 deficiency enhanced tumor growth in mice relative to that seen in controls. Single‐cell sequencing analysis of immune cells infiltrating tumors showed relatively high expression of S100a8/S100a9 in Tet2‐deficient myeloid subclusters. In turn, treatment with S100a8/S100a9 promoted Vegfa production by cancer cells, leading to a marked increase in the tumor vasculature in Tet2‐deficient mice relative to controls. Finally, treatment of Tet2‐deficient mice with an antibody against Emmprin, a known S100a8/S100a9 receptor, suppressed tumor growth. These data suggest that immune cells derived from TET2‐mutated clonal hematopoiesis exacerbate lung cancer progression by promoting tumor angiogenesis and may provide a novel therapeutic target for lung cancer patients with TET2‐mutated clonal hematopoiesis. Our study suggests that immune cells derived from TET2‐mutated clonal hematopoiesis exacerbate lung cancer progression by promoting tumor angiogenesis. We present evidence that signaling through the S100a8/S100a9‐Emmprin‐Vegfa axis is essential for progression of a lung cancer model established in a microenvironment of Tet2‐deficient immune cells. Furthermore, we provide a novel target for lung cancer patients with accompanying TET2‐mutated clonal hematopoiesis.

Smoking plays an underappreciated role in breast cancer progression, increasing recurrence and mortality in patients. Here, we show that S100A8/A9 innate immune signaling is a molecular mechanism that identifies smoking-related breast cancers and underlies their enhanced malignancy. In contrast to acute exposure, chronic nicotine increased tumorigenicity and reprogrammed breast cancer cells to express innate immune response genes. This required the α7 nicotinic acetylcholine receptor, which elicited dynamic changes in cell differentiation, proliferation, and expression of secreted cytokines, such as S100A8 and S100A9, as assessed by unbiased scRNA-seq. Indeed, pharmacologic or genetic inhibition of S100A8/A9-RAGE receptor signaling blocked nicotine’s tumor-promoting effects. We also discovered Syntaphilin ( SNPH ) as an S100A8/A9-dependent gene enriched specifically in estrogen receptor-negative (ER - ) cancers from former smokers, linking this response to patient disease. Together, our findings describe a new α7 nAChR-S100A8/A9-Syntaphilin immune signaling module that drives nicotine-induced tumor progression and distinguishes smoking-related patient disease as a distinct subset of aggressive breast cancers.

Myelodysplastic syndromes (MDS) are age-dependent stem cell malignancies that share biological features of activated adaptive immune response and ineffective hematopoiesis. Here we report that myeloid-derived suppressor cells (MDSC), which are classically linked to immunosuppression, inflammation, and cancer, were markedly expanded in the bone marrow of MDS patients and played a pathogenetic role in the development of ineffective hematopoiesis. These clonally distinct MDSC overproduce hematopoietic suppressive cytokines and function as potent apoptotic effectors targeting autologous hematopoietic progenitors. Using multiple transfected cell models, we found that MDSC expansion is driven by the interaction of the proinflammatory molecule S100A9 with CD33. These 2 proteins formed a functional ligand/receptor pair that recruited components to CD33’s immunoreceptor tyrosine-based inhibition motif (ITIM), inducing secretion of the suppressive cytokines IL-10 and TGF-β by immature myeloid cells. S100A9 transgenic mice displayed bone marrow accumulation of MDSC accompanied by development of progressive multilineage cytopenias and cytological dysplasia. Importantly, early forced maturation of MDSC by either all-trans-retinoic acid treatment or active immunoreceptor tyrosine-based activation motif–bearing (ITAM-bearing) adapter protein (DAP12) interruption of CD33 signaling rescued the hematologic phenotype. These findings indicate that primary bone marrow expansion of MDSC driven by the S100A9/CD33 pathway perturbs hematopoiesis and contributes to the development of MDS.

The production of chemoattractants in the pre-metastatic lung can be induced by distant primary tumours. The chemoattractants S100A8 and S100A9 induce serum amyloid A3 and TLR4 activation and cause an inflammation-like state that facilitates metastasis. A large number of macrophages and haematopoietic progenitor cells accumulate in pre-metastatic lungs 1 , 2 in which chemoattractants, such as S100A8 and S100A9, are produced by distant primary tumours serving as metastatic soil 3 . The exact mechanism by which these chemoattractants elicit cell accumulation is not known. Here, we show that serum amyloid A (SAA) 3, which is induced in pre-metastatic lungs by S100A8 and S100A9, has a role in the accumulation of myeloid cells and acts as a positive-feedback regulator for chemoattractant secretion. We also show that in lung endothelial cells and macrophages, Toll-like receptor (TLR) 4 acts as a functional receptor for SAA3 in the pre-metastatic phase. In our study, SAA3 stimulated NF-κB signalling in a TLR4-dependent manner and facilitated metastasis. This inflammation-like state accelerated the migration of primary tumour cells to lung tissues, but this was suppressed by the inhibition of either TLR4 or SAA3. Thus, blocking SAA3–TLR4 function in the pre-metastatic phase could prove to be an effective strategy for the prevention of pulmonary metastasis.

The protein heterodimer calprotectin and its component proteins, S100A8 and S100A9, play important antibacterial and pro-inflammatory roles in the mammalian innate immune response. Gaining mechanistic insights into the regulation and biological function of calprotectin will help facilitate patient diagnostics and therapy for inflammation and further our understanding of the host-microbe interface. Recent literature has identified zebrafish s100a10b as zebrafish calprotectin based on sequence similarity, genomic context, and transcriptional upregulation during the immune response to bacterial infections. The field would benefit from expanding the breadth of calprotectin studies into a zebrafish innate immunity model. Here, we carefully evaluated the possibility that zebrafish possess a calprotectin ortholog or a paralog that convergently evolved similar function. Using careful bioinformatics approaches, we found that zebrafish do not have an ortholog of either mammalian S100A8 or S100A9. To look for paralogs with convergent function, we identified four zebrafish s100 proteins—including s100a10b—that are expressed in immune cells and upregulated during the immune response. We recombinantly expressed and purified these proteins and measured their antimicrobial activity. None of the zebrafish proteins exhibited activity comparable to mammalian calprotectin. We also generated structural models of all homodimers and heterodimers of all annotated zebrafish s100 genes. None of these complexes were predicted to have an antimicrobial transition metal binding site equivalent to calprotectin. Finally, we measured the ability of our four purified zebrafish s100 proteins to activate inflammation via Toll-like receptor 4, a key feature of human S100A9; none of the proteins activated the receptor. Our work demonstrates conclusively that zebrafish have no ortholog of calprotectin and suggests that similar proteins have not convergently evolved analogous functions.

Renal tubulointerstitial damage is the final common pathway leading from chronic kidney disease to end-stage renal disease. Inflammation is clearly involved in tubulointerstitial injury, but it remains unclear how the inflammatory processes are initiated and regulated. Here, we have shown that in the mouse kidney, the transcription factor Krüppel-like factor-5 (KLF5) is mainly expressed in collecting duct epithelial cells and that Klf5 haploinsufficient mice (Klf5+/- mice) exhibit ameliorated renal injury in the unilateral ureteral obstruction (UUO) model of tubulointerstitial disease. Additionally, Klf5 haploinsufficiency reduced accumulation of CD11b+ F4/80(lo) cells, which expressed proinflammatory cytokines and induced apoptosis among renal epithelial cells, phenotypes indicative of M1-type macrophages. By contrast, it increased accumulation of CD11b+ F4/80(hi) macrophages, which expressed CD206 and CD301 and contributed to fibrosis, in part via TGF-β production--phenotypes indicative of M2-type macrophages. Interestingly, KLF5, in concert with C/EBPα, was found to induce expression of the chemotactic proteins S100A8 and S100A9, which recruited inflammatory monocytes to the kidneys and promoted their activation into M1-type macrophages. Finally, assessing the effects of bone marrow-specific Klf5 haploinsufficiency or collecting duct- or myeloid cell-specific Klf5 deletion confirmed that collecting duct expression of Klf5 is essential for inflammatory responses to UUO. Taken together, our results demonstrate that the renal collecting duct plays a pivotal role in the initiation and progression of tubulointerstitial inflammation.

Neutrophil accumulation is associated with lung pathology during active tuberculosis (ATB). However, the molecular mechanism or mechanisms by which neutrophils accumulate in the lung and contribute to TB immunopathology are not fully delineated. Using the well-established mouse model of TB, our new data provide evidence that the alarmin S100A8/A9 mediates neutrophil accumulation during progression to chronic TB. Depletion of neutrophils or S100A8/A9 deficiency resulted in improved Mycobacterium tuberculosis (Mtb) control during chronic but not acute TB. Mechanistically, we demonstrate that, following Mtb infection, S100A8/A9 expression is required for upregulation of the integrin molecule CD11b specifically on neutrophils, mediating their accumulation during chronic TB disease. These findings are further substantiated by increased expression of S100A8 and S100A9 mRNA in whole blood in human TB progressors when compared with nonprogressors and rapidly decreased S100A8/A9 protein levels in the serum upon TB treatment. Furthermore, we demonstrate that S100A8/A9 serum levels along with chemokines are useful in distinguishing between ATB and asymptomatic Mtb-infected latent individuals. Thus, our results support targeting S100A8/A9 pathways as host-directed therapy for TB.